Long-persistence homogeneous detection material based on reference calibration, detection method and system

Abstract

The application discloses a long-afterglow homogeneous phase detection material based on reference calibration, which comprises a long-afterglow detection system, and the long-afterglow detection system is used for detecting a to-be-detected object and comprises a donor label and an acceptor label, wherein the donor label comprises a sensitizing agent, the acceptor label buffer agent and a luminescent agent; the material further comprises a reference substance which is used for being introduced into the donor label and / or the acceptor label, and calibrating the detection result of the to-be-detected object; the long-afterglow detection system does not have energy transfer with the reference substance, and there is a wavelength difference between the luminescent wavelength of the reference substance and the luminescent wavelength of the long-afterglow detection system. The application further discloses a corresponding detection method and a detection system; when a kind of reference substance is introduced into the donor label and / or the acceptor label, the calibration of environmental factors and instrument equipment factors can be realized; when the long-afterglow is used for calibrating the method of immunodetection based on a FRET luminescent system, the calibration of non-immune binding false signals can be realized.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay and relates to a long-persistence homogeneous detection material, detection method and system based on reference calibration. Background Technology

[0002] Long-persistent homogeneous detection is a homogeneous assay method applicable to immunoassay. 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 concentration of the analyte. 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 an energy transfer process, 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, and the effective range is approximately 100 nm. By detecting the intensity of the long-persistent luminescence signal, the presence and concentration of the analyte in the sample can be determined. This method effectively avoids cumbersome steps such as elution and separation. 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 greatly improving detection efficiency and cost-effectiveness. Furthermore, using long-persistent luminescence as the detection signal effectively avoids interference from autofluorescence, thus achieving a high signal-to-noise ratio.

[0003] However, current detection methods generally use single luminescence intensity information as the basis for immunoassay. This detection method is easily affected by external environmental factors and cannot exclude false signals generated by the binding of donor and recipient microspheres due to non-immune reactions, thus making it difficult to obtain accurate detection results. Summary of the Invention

[0004] To address the above problems, this invention discloses a long-persistence homogeneous material, detection method, and system based on reference calibration. The aim is to propose a long-persistence homogeneous material detection method that calibrates the detection results by introducing an intrinsic reference system, and simultaneously create a long-persistence homogeneous material detection system based on this method.

[0005] One of the objectives of this invention is to provide a long-persistence homogeneous detection material for reference calibration, which is based on the long-persistence luminescent reagent (referred to as the long-persistence detection system in this invention) disclosed in invention patent CN202010347677.8. It includes a donor marker and an acceptor marker, wherein the donor marker contains a sensitizer and the acceptor marker contains a buffer and a luminescent agent.

[0006] Donor markers generally refer to the counterparts that are attached to a photochemical energy donor and can specifically bind to the substance to be detected; the photochemical energy donor includes the sensitizer described in this invention, which can generate singlet oxygen under photoexcitation;

[0007] Receptor markers generally refer to another counterpart attached to a photochemical energy acceptor that can specifically bind to the substance to be detected; photochemical energy acceptors include the buffer and luminescent agent described in this invention, wherein the energy acceptor can react with singlet oxygen to produce afterglow luminescence signal.

[0008] In this application, photochemical energy donor and FRET energy donor are different terms; photochemical energy acceptor and FRET energy acceptor are different terms.

[0009] More preferably, the photochemical energy donor is selected from donor microspheres formed using nanospheres as a carrier matrix for sensitizing agents, and the photochemical energy acceptor is selected from acceptor microspheres formed using nanospheres as a carrier matrix for both buffering and luminescent agents.

[0010] The present invention aims to calibrate the test results of homogeneous detection of long afterglow systems described in the present invention and disclosed by the inventors by introducing a reference material.

[0011] The following example, using donor microspheres and acceptor microspheres as photochemical energy donors and acceptors respectively, explains the principle of long-afterglow homogeneous detection. Figure 1 As shown, the detection method is based on the effective energy transfer effect generated by the proximity (10nm~100nm) between the donor and acceptor markers, thereby utilizing this effect to homogeneously detect the analyte. In this method, a photoexcited photochemical energy donor (donor microspheres, for example, those encapsulated with phthalocyanine molecules) is typically used, while the photochemical energy acceptor, i.e., the acceptor microsphere, approaches the donor microsphere through a specific immunoreaction. Through the energy transfer process, a long-afterglow emission of a certain wavelength is emitted. The presence of the analyte in the actual sample is then determined by detecting the intensity of the long-afterglow emission signal.

[0012] The present invention aims to add reference substances to donor markers and / or recipient markers to eliminate interference from the environment, instruments, or the detection method itself, thereby further improving the detection accuracy of long afterglow materials in homogeneous immunoassay.

[0013] The following is a specific embodiment of the present invention:

[0014] Reference-calibrated long-persistence homogeneous detection materials include a long-persistence detection system for detecting analytes, including donor markers and acceptor markers, wherein the donor markers contain sensitizers and the acceptor markers contain buffers and luminescent agents;

[0015] It also includes reference materials, which are introduced into the long afterglow detection system to calibrate the detection results of the analyte;

[0016] The long-afterglow detection system does not transfer energy to the reference material, and the emission wavelength and / or emission lifetime of the reference material differ from those of the long-afterglow detection system.

[0017] Preferably, the reference substance is introduced into the donor marker and / or the acceptor marker.

[0018] Preferably, the reference material is introduced into the donor and / or acceptor markers through chemical bonding, charge adsorption, or encapsulation.

[0019] Preferably, the difference between the emission wavelength of the reference material and the emission wavelength of the long-afterglow detection system is greater than 10 nm. Maintaining a difference of more than 10 nm in the emission wavelength is more effective in improving the accuracy of the detection results.

[0020] Further preferred, the difference in emission wavelength is greater than 20 nm, and more preferably, the difference in emission wavelength is greater than 30 nm.

[0021] In addition, donor markers and / or acceptor markers, and reference substances can be coated on the outside of the microspheres or encapsulated / embedded within the microspheres, and can be selectively introduced into the donor microspheres and / or acceptor microspheres. The carrier matrix in the donor and acceptor microspheres can be the carrier microspheres disclosed in CN202010347677.8, and the microsphere particle size range is preferably 30 nm to 1000 nm, more preferably 100 nm to 500 nm.

[0022] The sensitizer, buffer, and luminescent agent can be selected from the light absorber, photochemical buffer, and luminescent agent disclosed in CN202010347677.8, respectively.

[0023] Preferably, when the reference material is introduced into the donor label, the absorption energy level of the reference material does not overlap with the emission energy level of the sensitizer, and the wavelengths corresponding to the energy levels differ by more than 10 nm, preferably by more than 30 nm, and more preferably by more than 50 nm.

[0024] When the reference material is introduced into the acceptor label, the absorption energy level of the reference material does not overlap with the emission energy level of the luminescent agent, and the wavelengths corresponding to the energy levels differ by more than 10 nm, preferably by more than 30 nm, and more preferably by more than 50 nm.

[0025] When the reference material is introduced into the donor marker and the donor marker, the absorption energy level of the reference material does not overlap with the emission energy levels of the sensitizer, buffer and luminescent agent, and the wavelengths corresponding to the energy levels differ by more than 10 nm, preferably more than 30 nm, and more preferably more than 50 nm.

[0026] Preferably, the reference material can be selected from one of the following: fluorescent material, upconversion luminescence system, room temperature phosphorescence system, and organic long afterglow system.

[0027] Preferably, the reference substance can be selected from one of the following: cyanine fluorescent dyes, rhodamine fluorescent dyes, fluoroboron dipyrrole fluorescent dyes, rare earth nanomaterials, luminescent complexes, or luminescent polymers.

[0028] Preferably, the reference material can be one of the following: a self-luminescent temperature probe (Chem. Soc. Rev., 2013, 42, 7834–7869.), a luminescent oxygen probe (Chem. Soc. Rev., 2010, 39, 3102–3114.), or a laser dye (Chem. Rev., 2007, 107, 1272-1295.).

[0029] More specifically, selectable luminescent temperature probes can be used to calibrate test results by monitoring the temperature of the immune response system;

[0030] A luminescent oxygen probe can be used to monitor the oxygen concentration or pressure at the site of the immune response to calibrate the test results;

[0031] Laser dyes can be used to monitor the intensity of the excitation light to calibrate the test results.

[0032] Preferably, the sensitizer is selected from dyes or quantum dots;

[0033] Alternatively, the sensitizer may be selected from donor microspheres formed by introducing them into nanospheres;

[0034] The caching agent and the luminescent agent are linked by chemical bonds, or the caching agent and the luminescent agent are introduced into the nanospheres to form receptor microspheres.

[0035] Specifically, sensitizers, such as dyes or quantum dots, are directly linked to proteins to prepare donor markers, which are non-particle-based. Quantum dots are introduced into nanospheres to form donor microspheres, and proteins are then linked to these microspheres as donor markers, which are particle-based. Buffers and luminescent agents are linked to proteins directly through chemical bonds to prepare receptor markers, which are non-particle-based. Buffers and luminescent agents are introduced into nanospheres to form receptor microspheres, and proteins are then linked to these microspheres as receptor markers, which are particle-based.

[0036] Preferably, the molar ratio of sensitizer to reference substance is 1000:1 to 1:1; more preferably, the molar ratio is 100:1 to 1:1.

[0037] Specifically, the proportions of the sensitizer, luminescent agent, and buffer have been disclosed by the inventors in other literature. The molar ratio of the sensitizer to the reference substance is 1000:1 to 1:1; preferably, the molar ratio is 100:1 to 1:1, more preferably, 100:1 to 10:1. The ratio can be selected and optimized according to experimental needs.

[0038] Preferably, the reference substance includes: a first reference molecule and a second reference molecule;

[0039] The reference-calibrated long-persistence homogeneous detection system can be applied to homogeneous immunoassay of the materials in the above scheme, realizing photoexcitation, signal collection, and data analysis and processing of the reference material and the long-persistence detection system, including:

[0040] A sample chamber configured to hold a homogeneous immunoassay system to be tested;

[0041] A light source device configured to output excitation light for exciting a long-afterglow homogeneous detection material containing a reference substance to illuminate the sample chamber;

[0042] A light acquisition device configured to acquire light from the sample chamber;

[0043] A detection device configured to receive light output from the acquisition device and generate a spectrum or kinetic curve based on the received light; and

[0044] A processing device configured for data processing.

[0045] Preferably, the reference-calibrated long-afterglow homogeneous detection method, using the reference-calibrated long-afterglow homogeneous detection material described in the above scheme, and the detection system described in the above scheme, includes the following steps:

[0046] S1. Provides a standard curve of the optical signal of the parameter to be calibrated and the standard curve of the concentration of the long afterglow light signal of the parameter to be calibrated and the analyte.

[0047] S2. Provide long afterglow homogeneous detection materials containing reference substances;

[0048] S3. The sample to be tested, diluent, and long afterglow homogeneous detection material are added to the reaction cup in sequence and stirred evenly to prepare an immunoreaction system. Then the reaction cup is placed in the sample chamber.

[0049] S4. Turn on the light source device and collect the light signal F1 of the reference material and the light signal F2 of the long afterglow system material by the collection device.

[0050] S5. The processing device processes the data, substitutes F1 into the standard curve of the parameter to be calibrated - the reference substance light signal, and obtains the value of the parameter to be calibrated. The obtained value of the parameter to be calibrated and F2 are then substituted into the standard curve cluster of the parameter to be calibrated - long afterglow light signal - concentration of the analyte to obtain the concentration of the analyte.

[0051] Preferably, the parameters to be calibrated include environmental factors or instrument factors. Among them, environmental factors include temperature or oxygen. When the factor to be calibrated is temperature, a luminescent temperature probe can be used; when the factor to be calibrated is oxygen or air pressure, a luminescent oxygen probe can be used; when the factor to be calibrated is excitation light intensity, an excitation dye can be used.

[0052] In the FRET system, an energy transfer phenomenon occurs between two closely spaced fluorescent molecules. The emission energy level of the donor fluorescent molecule overlaps with the absorption energy level of the acceptor fluorescent molecule, and this process occurs when the distance between the two molecules is within 10 nm. When the distance between the donor and acceptor fluorescent molecules is less than 10 nm, the system exhibits the characteristic luminescence of the acceptor fluorescent molecule.

[0053] Taking particle type as an example,

[0054] like Figure 1 As shown, in the ideal state, without the analyte bound, the distance between the donor and acceptor microspheres is greater than 100 nm, making it difficult for singlet oxygen to effectively transfer energy. Therefore, no long-afterglow luminescence signal is generated. However, in actual detection... Figure 2 Due to the free diffusion of microspheres or physical adsorption and binding, the distance between donor microspheres and acceptor microspheres may be less than 10 nm, or even in zero-distance contact, which can still result in efficient long-afterglow luminescence and thus generate pseudo signals.

[0055] The inventors aim to remove spurious signals and make the detection results more accurate by introducing a reference substance into the long afterglow homogeneous detection material.

[0056] like Figure 3The calibration principle is explained as follows: A reference substance is introduced into the microspheres. This can be either a first or second reference molecule in the donor microspheres, or a second or first reference molecule in the acceptor microspheres. The first and second reference molecules form a FRET system. The emission wavelength of the reference substance differs from that of the long-persistence detection system, allowing for differentiation based on wavelength. Alternatively, the long lifetime of the long-persistence system can be utilized to differentiate the emission signals of the reference substance and the long-persistence detection system using time-resolved technology. In normal immune binding, the distance between the donor and acceptor microspheres is greater than 10 nm, preventing FRET emission. Therefore, the presence of FRET emission indicates an abnormal distance between the donor and acceptor microspheres. Subsequent anomaly correction is performed, calibrating the emission signal / intensity to improve the accuracy of the detection results.

[0057] The specific plan is as follows:

[0058] The present invention also provides a long-afterglow homogeneous detection material based on reference calibration, wherein the reference substance includes a first reference molecule and a second reference molecule;

[0059] The first and second reference molecules are introduced into the donor and acceptor markers, respectively, and the first and second reference molecules constitute a fluorescence resonance energy transfer (FRET) luminescence system.

[0060] When the first reference molecule is a FRET donor molecule, the second reference molecule is a FRET acceptor molecule; when the first reference molecule is a FRET acceptor molecule, the second reference molecule is a FRET donor molecule.

[0061] Preferably, the wavelength difference between the emission wavelength of the reference material and the emission wavelength of the long-persistence detection system is greater than 10 nm; more preferably, the wavelength difference is greater than 20 nm, and even more preferably, the wavelength difference is greater than 30 nm. Maintaining a wavelength difference greater than 10 nm is better for improving the accuracy of the detection results, and a larger wavelength difference is more conducive to improving the accuracy of the detection results. Preferably, the difference is 30 nm, 50 nm, or 100 nm.

[0062] Preferably, when the first reference molecule is the energy donor of the FRET system, the second reference molecule is the energy acceptor of the FRET system; when the second reference molecule is the energy donor of the FRET system, the first reference molecule is the energy acceptor of the FRET system.

[0063] When the first reference molecule is introduced into the donor label, the absorption energy level of the first reference molecule does not overlap with the emission energy level of the sensitizer, and the wavelengths corresponding to the energy levels differ by more than 10 nm, preferably by more than 30 nm, and more preferably by more than 50 nm.

[0064] When the second reference molecule is introduced into the receptor label, the absorption energy level of the second reference molecule does not overlap with the emission energy levels of the buffer and the luminescent agent, and the wavelengths corresponding to the energy levels differ by more than 10 nm, preferably by more than 30 nm, and more preferably by more than 50 nm.

[0065] Preferably, the first reference molecule and the second reference molecule are selected from one of the following: fluorescent substances, upconversion luminescence systems, room temperature phosphorescence systems, and organic long afterglow systems.

[0066] When distinguishing between the luminescence of a reference material and a long-persistent luminescence system, the luminescence signals at their respective wavelengths can be collected separately. Additionally, the afterglow of the long-persistent luminescence system can be used for time-resolved differentiation, which can also achieve the removal of spurious signals.

[0067] Preferably, the molar ratio of the first reference molecule to the sensitizer is 1000:1 to 1:1, and more preferably, the molar ratio is 100:1 to 1:1.

[0068] The molar ratio of the first reference molecule to the second reference molecule is 1:3 to 3:1, with a preferred molar ratio of 1:1.

[0069] The materials described in the above scheme can also use the aforementioned reference-calibrated long-afterglow homogeneous detection system to achieve photoexcitation, signal collection, and data analysis and processing of the reference material and the long-afterglow detection system.

[0070] This invention also discloses a long-persistence homogeneous detection method based on reference calibration, providing the aforementioned long-persistence homogeneous detection material based on reference calibration, and employing the aforementioned detection system, comprising the following steps:

[0071] S100 provides standard curves for anomaly rate versus reference material optical signal and standard curves for anomaly rate versus long-persistence optical signal.

[0072] S200 provides a long afterglow signal-analyte concentration standard curve;

[0073] S300, providing long afterglow homogeneous detection materials containing reference substances;

[0074] S400. The sample to be tested, diluent, and long afterglow homogeneous detection material are added to the reaction cup in sequence and stirred evenly to prepare an immunoreaction system. Then the reaction cup is placed in the sample chamber.

[0075] S500, turn on the light source device, and the acquisition device acquires the light signal F1 of the reference material and the light signal F2 of the long afterglow system material;

[0076] S600. The processing device processes F1 and substitutes it into the standard curve of anomaly rate-reference material light signal to obtain the anomaly rate. The obtained anomaly rate is then substituted into the standard curve of anomaly rate-long afterglow light signal to obtain the long afterglow light signal F3. Finally, F2-F3 are substituted into the standard curve of long afterglow light signal-detection substance concentration to obtain the detection concentration.

[0077] The steps for obtaining the standard curves of the anomaly rate versus the reference material optical signal and the anomaly rate versus the long-persistence optical signal are as follows:

[0078] S1001, Component 1 consists of donor microspheres and acceptor microspheres containing a reference substance connected by chemical bonds.

[0079] The long afterglow detection system was used as component 2.

[0080] S1002. Component 1 and component 2 are allocated in N different proportions to form N abnormal groups, which are denoted as N different abnormal rates. Component 1 and component 2 of the N abnormal groups are added to the reaction system to be tested, and the above detection system is used to test them to obtain N reference material light signals F01 and N long afterglow light signals F02.

[0081] The standard curve of the anomalous rate versus the reference material optical signal is obtained by fitting S1003, N F01 and N different anomalous rates; the standard curve of the anomalous rate versus the long afterglow optical signal is obtained by fitting N F02 and N different anomalous rates.

[0082] The steps for obtaining the long afterglow signal-analyte concentration standard curve are as follows:

[0083] S2001, Provides M different concentrations of the substance to be detected.

[0084] S2002. Add the long-afterglow homogeneous detection material containing the reference material to the analyte and test it using the above detection system to obtain M reference material light signals F011 and M long-afterglow light signals F022.

[0085] S2003. Substitute the M reference substance light signals F011 into the standard curve of anomaly rate-reference substance light signal to obtain the anomaly rate. Substitute the obtained anomaly rate into the standard curve of anomaly rate-long afterglow light signal to obtain M long afterglow light signals F033, F022-F033 and M kinds of analyte concentrations to obtain the standard curve of long afterglow light signal-analyte concentration.

[0086] Where N and M are integers greater than 3.

[0087] In summary, by introducing a reference substance into the long-afterglow homogeneous detection material, this invention can effectively eliminate the influence of external environmental factors and calibrate the spurious signals generated by the non-immune binding of donor and acceptor microspheres, thereby obtaining accurate detection results. Attached Figure Description

[0088] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely explanations of the principles or embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0089] Figure 1 The attached diagram illustrates the principle of long afterglow homogeneous detection.

[0090] When the analyte is not immunobound, the distance between the donor microsphere and the recipient microsphere is >100nm, making it difficult to transfer energy effectively and resulting in no long-lasting luminescence. When the analyte is immunobound, the distance between the donor microsphere and the recipient microsphere is 10-100nm, which enables effective energy transfer and results in long-lasting luminescence.

[0091] Figure 2 The attached diagram illustrates the generation of pseudo-signals in long-persistence homogeneous detection.

[0092] The left image shows efficient short-range energy transfer caused by free diffusion approach;

[0093] The right figure shows the ultra-efficient zero-distance energy transfer caused by physical adsorption and binding;

[0094] Figure 3 The attached figure is a schematic diagram of pseudo-signal correction and reference calibration for long-persistence homogeneous detection;

[0095] Figure i shows the correction for free diffusion approaching the spurious signal;

[0096] Figure ii shows the correction for the pseudo-signal of physical adsorption binding;

[0097] Figure 4 The attached figure shows the long afterglow emission spectrum of the europium complex luminescent agent in Example 1;

[0098] Figure 5 The attached figure shows the upconversion emission spectrum of the upconversion luminescent material in Example 1;

[0099] Where * represents excitation light;

[0100] Figure 6 The attached figure shows the temperature-light signal intensity curve of the upconversion luminescent material in Example 1;

[0101] Figure 7 The attached figure shows the standard curve cluster of long afterglow signal-analyte concentration in Example 1;

[0102] Figure 8 The attached figure shows the concentration of the analyte and the long-afterglow luminescence intensity before and after calibration in Example 2.

[0103] Figure labels: 1. Donor microsphere; 2. Receptor microsphere; 3. Correspondent 1 that specifically binds to the biomarker; 4. Correspondent 2 that specifically binds to the biomarker; 5. Excitation by a light source; 6. Emission without long afterglow; 7. Emission with long afterglow; 8. Detector; 9. First reference molecule; 10. Second reference molecule. Detailed Implementation

[0104] Example 1

[0105] The reference-calibrated long-persistence homogeneous detection material, system, and homogeneous detection method of this invention were used to test C-reactive protein (CRP) in whole blood samples.

[0106] 1.1 Materials and Equipment Used

[0107] Test samples: 25 random samples were collected at one time, including whole blood samples from normal people and whole blood samples from patients with inflammation;

[0108] Long afterglow detection system:

[0109] Sensitizer: Phthalocyanine dye, excitation wavelength 730nm;

[0110]

[0111] Luminescent agent: europium complex, emission wavelength 615 nm; spectrum shown in Figure 4;

[0112]

[0113] Cache:

[0114]

[0115] Reference material: Upconversion luminescent material (temperature probe), excitation: 730 nm, emission: 660 nm, spectrum as follows Figure 5 As shown;

[0116]

[0117] Corresponding antibodies that specifically bind to the biomarker CRP: CRP-Ab1 monoclonal antibody, CRP-Ab2 monoclonal antibody;

[0118] Carrier matrix: Carboxylated polystyrene spheres (nanosphere type);

[0119] The detection system includes a sample chamber, a light source, a data acquisition device, a detection device, and a processing device.

[0120] The sample chamber is configured to hold the homogeneous immunoassay system to be tested;

[0121] The light source is configured to output excitation light to stimulate a long-afterglow homogeneous detection material containing a reference material to illuminate the sample chamber.

[0122] The acquisition device is configured to acquire light from the sample chamber;

[0123] The detection device is configured to receive light output from the acquisition device and generate a spectrum or kinetic curve based on the received light;

[0124] The processing unit is configured for data processing.

[0125] 1.2 Preparation of donor microspheres with reference material and coating with CRP-Ab1 monoclonal antibody

[0126] Preparation of donor microspheres with reference material:

[0127] Dissolve 0.1g of carboxylated polystyrene spheres with a particle size of 100nm in 100mL of ultrapure water and sonicate to form a dispersed phase; add 0.5mL to 5mL each of 2wt% sodium dodecylbenzenesulfonate and 1wt% ethylenediamine polyoxyethylene polyoxypropylene block polyether to the dispersion and stir to obtain an aqueous phase;

[0128] The reference substance and sensitizer were dispersed in 10 mL of tetrahydrofuran solution at a molar ratio of 1:1. After the solution was prepared, it was quickly added to the above aqueous phase. The temperature was then gradually increased to 50 °C, and the mixture was stirred continuously for 10 h before centrifugation. The obtained carboxylated polystyrene spheres were washed twice with ultrapure water and ethanol and stored in ultrapure water to prepare donor microspheres. These microspheres were then stored at room temperature in the dark for later use.

[0129] Corresponding agents that specifically bind to biomarkers:

[0130] The donor microspheres prepared above were centrifuged at high speed, washed three times with BBS buffer, and finally adjusted to a solid content of 0.5%. They were then ultrasonically dispersed to obtain a dispersion.

[0131] Add 500 μL of the above dispersion to 500 μL of MES and 10 mg of EDC, and react at room temperature for 2 hours. After the reaction, centrifuge and wash, then reconstitute each mixture into 500 μL of PBS buffer. Add 0.05 mg of CRP-Ab1 monoclonal antibody to each buffer and react at room temperature for 8 hours. After the reaction, centrifuge and wash at 35,000 rpm, then reconstitute into 500 μL of PBS buffer. Add 10 mg of BSA to each buffer and react at room temperature for 8 hours. After the reaction, centrifuge and wash to obtain donor microspheres coated with CRP-Ab1 antibody. Reconstitute these microspheres into 1.0 mL of BBS buffer and store at 4 °C for later use.

[0132] 1.3 Preparation of receptor microspheres and coating with CRP-Ab2 monoclonal antibody

[0133] Acceptor microspheres were prepared according to the method for preparing donor microspheres in 1.2, wherein the luminescent agent and the buffer were dispersed in 10 mL of tetrahydrofuran solution instead of the sensitizer and the reference substance.

[0134] CRP-Ab2 monoclonal antibody was conjugated onto the receptor microspheres according to the conjugation method in 1.2 to obtain receptor microspheres coated with CRP-Ab2 antibody. These microspheres were then reconstituted in 1.0 mL of BBS buffer and stored at 4 °C for later use.

[0135] 1.4 Preparation of the reagent kit

[0136] The microsphere protectant was prepared with the following composition: 20 μL 20% (w / v) glucose, 20 μL 10% (w / v) mannitol, 20 μL 0.5% (w / v) BSA and 20 μL 0.01 (v / v) Proclin-300. The donor microspheres coated with CRP-Ab1 antibody prepared in step 1.2 and the recipient microspheres coated with CRP-Ab2 antibody prepared in step 1.3 were diluted to a solid content of 0.01% using the microsphere protectant described above. Then, they were mixed separately at a 1:1 volume ratio with 100 mM BBS buffer (prepared with purified water, containing 0.5% (w / v) PEG, 0.1% (w / v) SDS, 0.9% (w / v) NaCl, 0.1% (w / v) KCl, 0.5% (w / v) BSA, pH 7.0–pH 7.6). The mixture was then dispensed into reagent bottle 1 (Reagent 1) and reagent bottle 2 (Reagent 2) to obtain the finished kit. The finished kit should be stored at 2–8°C protected from light.

[0137] 1.5 Detection Method

[0138] Detecting CRP in whole blood samples

[0139] 1.51 Establishing a standard curve for temperature-reference material optical signal

[0140] like Figure 6 As shown, the temperature-light signal intensity curve of the upconversion luminescent material.

[0141] 1.52 Establish a standard curve for temperature gradient, long afterglow signal, and analyte concentration.

[0142] The system temperature is maintained at 10℃.

[0143] 1) Dilute Yu Hui's biomarker CRP antigen into six CRP antigen dilutions with progressively increasing concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 300 mg / L, and 400 mg / L using a diluent (50 mM PBS buffer containing 1% (w / v) BSA, 250 mM sodium chloride, and 0.1% (w / v) preservative).

[0144] 2) Add 2 μL of one of the above CRP antigen diluents, 98 μL of diluent, 150 μL of reagent 1, and 150 μL of reagent 2 to the same reaction vessel in sequence, stir well to prepare an immune reaction system, and incubate for 5 min;

[0145] 3) Irradiate with 730nm excitation light, and collect the reference substance light signal value F1; after 10ms, collect the long afterglow light signal value F2, and obtain the standard curve of 10℃-long afterglow light signal-analyte concentration by fitting.

[0146] 1.53 The system temperature was controlled at 25℃ and 40℃ respectively, and steps 1), 2), and 3) were repeated to obtain multiple long-persistent glow signal values ​​F2. Standard curves at 10℃, 25℃, and 40℃, based on the long-persistent glow signal and the concentration of the analyte, were obtained through fitting, as shown below. Figure 7 As shown.

[0147] 1.54 Detection of the biomarker CRP in the test sample (whole blood)

[0148] 1) Add 2 μL of the whole blood sample to be tested, 98 μL of diluent, 150 μL of reagent 1 and 150 μL of reagent 2 to the same reaction vessel in sequence, stir well to prepare an immunoreaction system, and incubate for 5 min;

[0149] 2) Irradiate the reaction cup with 730nm excitation light, collect the reference material light signal value F11, turn off the excitation light, and collect the long afterglow signal value F22 after 10ms.

[0150] 3) Substitute F11 into the standard curve of temperature-reference material light signal to obtain the temperature value. Substitute the temperature value into the standard curve cluster of 10℃, 25℃, and 40℃-long afterglow light signal-analyte concentration to obtain the analyte concentration. When the temperature is between two standard curves of long afterglow light signal-analyte concentration, use interpolation to obtain the standard curve of long afterglow light signal-analyte concentration at that temperature and then calculate the analyte concentration.

[0151] Typically, experimental data are directly substituted into a standard curve (e.g., a standard curve established at room temperature of 25°C) without considering calibration to calculate results. However, the actual temperature during testing may deviate from 25°C, leading to inaccurate results. After calibrating the temperature using a reference, the obtained analyte concentration is closer to the reference value measured by medical institutions (as shown in Table 1).

[0152] Table 1

[0153]

[0154] Note: A / B / C represent the detected concentration values. A: Before reference calibration (mg / L) B: After reference calibration (mg / L) C: Concentration reference value (mg / L)

[0155] Example 2: Removal of Non-Immune Binding Spurious Signals

[0156] 2.1 Materials and Equipment Used

[0157] Test samples: 10 random samples were collected at a time, including whole blood samples from healthy individuals and whole blood samples from patients with inflammation.

[0158] Long afterglow detection system:

[0159] Sensitizer: platinum benzo[a]porphyrin complex, excitation wavelength 610 nm;

[0160]

[0161] Luminescent agent: Perylene, emission wavelength 450nm;

[0162]

[0163] Cache:

[0164]

[0165] Reference material:

[0166] First reference molecule: absorbs excitation light at 610 nm and emits fluorescence at 670 nm;

[0167]

[0168] Second reference molecule: absorbs light at 670nm and emits fluorescence at 700nm;

[0169]

[0170] Corresponding antibodies that specifically bind to biomarkers: CRP-Ab1 monoclonal antibody, CRP-Ab2 monoclonal antibody;

[0171] Carrier matrix: Carboxylated polystyrene spheres (nanosphere type);

[0172] The detection system includes a sample chamber, a light source, a data acquisition device, a detection device, and a processing device.

[0173] The sample chamber is configured to hold the homogeneous immunoassay system to be tested, and in this embodiment, the temperature is controlled to be fixed at 37°C.

[0174] The light source is configured to output excitation light to stimulate a long-afterglow homogeneous detection material containing a reference material to illuminate the sample chamber.

[0175] The acquisition device is configured to acquire light from the sample chamber;

[0176] The detection device is configured to receive light output from the acquisition device and generate a spectrum or kinetic curve based on the received light;

[0177] The processing unit is configured for data processing.

[0178] 2.2 Preparation of donor microspheres containing the first reference molecule X1 and coating them with CRP-Ab1 monoclonal antibody

[0179] The preparation steps are similar to those in step 1.2, except that the reference substance is replaced with X1, resulting in donor microspheres with X1 introduced and donor microspheres with X1 introduced and coated with CRP-Ab1 antibody.

[0180] 2.3 Preparation of receptor microspheres with the introduction of the second reference molecule X2 and coating them with CRP-Ab1 monoclonal antibody

[0181] The preparation steps are similar to those in step 1.3, except that X2 is added as a reference substance to obtain receptor microspheres with X2 introduced and receptor microspheres with X2 introduced and coated with CRP-Ab2 antibody.

[0182] 2.4 Following the method in step 1.4, prepare the donor microspheres containing X1 and coated with CRP-Ab1 antibody as reagent 3, and prepare the recipient microspheres containing X2 and coated with CRP-Ab2 antibody as reagent 4.

[0183] The molar ratio of X1 to X2 is 1:1.

[0184] 2.5 Preparation of systems with abnormality rates of 1%, 5%, 10%, 15%, 20%, 25%, and 30% respectively:

[0185] The donor microspheres containing X1 and the acceptor microspheres containing X2 are connected by chemical bonds (bonding groups, such as carboxyl and hydroxyl groups, are attached to the surface of the microspheres) and are called component 1, wherein the amounts of the two types of microspheres are equal;

[0186] The donor microspheres containing X1 and the acceptor microspheres containing X2 are directly mixed and referred to as component 2, with equal amounts of both types of microspheres used. The anomaly rate is defined by the concentration ratio of component 1 to component 2 microspheres. Taking a 10% anomaly rate as an example, the concentration ratio of component 1 to component 2 is 1:9.

[0187] 2.6 Detection Method

[0188] 2.61 Establish standard curves for anomaly rate versus reference material optical signal and anomaly rate versus long-persistence optical signal.

[0189] Systems with abnormality rates of 1%, 5%, 10%, 15%, 20%, 25%, and 30% (300 μL) were prepared using 2.5 and added to 100 μL of dilution buffer (50 mM PBS buffer containing 1% BSA, 250 mM sodium chloride, and 0.1% preservative) (antigen may or may not be added).

[0190] 2) Irradiate with 610nm excitation light to obtain multiple reference material light signal values ​​F01 and multiple long-persistence light signal values ​​F02. The standard curves of anomaly rate-reference material light signal and anomaly rate-long-persistence light signal are obtained by fitting.

[0191] 2.62 Establish a standard curve for long-persistent light signal-analyte concentration.

[0192] 1) Dilute the CRP antigen to the following concentrations using diluent: 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 300 mg / L, and 400 mg / L, with the diluent used as 0 mg / mL.

[0193] 2) Add 2 μL of one of the above CRP antigen diluents, 98 μL of diluent, 150 μL of reagent 3, and 150 μL of reagent 4 (molar ratio of the first reference molecule to the second reference molecule is 1:1) to the same reaction vessel in sequence, stir well to prepare an immune reaction system, and incubate for 5 min;

[0194] 3) Irradiate with 610nm excitation light to obtain multiple reference material light signal values ​​F011; turn off the excitation light, and after 10ms, obtain multiple long-persistence light signal values ​​F022. By fitting, substitute the multiple reference material light signals F011 into the standard curve of anomaly rate - reference material light signal to obtain the anomaly rate. Substitute the obtained anomaly rate into the standard curve of anomaly rate - long-persistence light signal to obtain multiple long-persistence light signals F033. Fit multiple (F022-F033) values ​​and multiple CRP concentration values ​​to obtain the standard curve of long-persistence light signal - analyte concentration.

[0195] 2.63. Detection of the biomarker CRP in the test sample (whole blood)

[0196] 1) Add 2 μL of the whole blood sample to be tested, 98 μL of diluent, 150 μL of reagent 3 and 150 μL of reagent 4 to the same reaction vessel in sequence, stir well to prepare an immunoreaction system, and incubate for 5 min;

[0197] 2) Irradiate the reaction cup with 610nm excitation light, collect the reference material light signal value F011”, turn off the excitation light, and collect the long afterglow signal value F022” after 10ms;

[0198] 3) Substitute F011 into the standard curve of anomaly rate-reference material light signal to obtain the anomaly rate. Substitute the obtained anomaly rate into the standard curve of anomaly rate-long afterglow light signal to obtain the afterglow signal F033. Substitute (F022”-F033”) into the standard curve of long afterglow light signal-analyte concentration to obtain the analyte concentration.

[0199] Typically, experimental data are calculated directly by substituting it into an uncalibrated standard curve without considering calibration. However, actual testing conditions may differ (e.g., different abnormal states caused by non-immune binding), leading to inaccurate results. Figure 8 As shown in the figure, the graph of multiple long-persistent signal values ​​F022”, multiple corrected values ​​(F022”-F033”) and gradient concentrations shows that this method can effectively correct spurious signals. After calibrating the anomaly rate by reference, the obtained concentration of the analyte is closer to the concentration reference value measured by medical institutions (as shown in Table 2).

[0200] Table 2

[0201]

[0202] Note: A / B / C represent the detected concentration values. A: Before reference calibration (mg / L) B: After reference calibration (mg / L) C: Concentration reference value (mg / L)

[0203] Example 3

[0204] Example 1 was repeated, except that a reference substance was introduced into the receptor microspheres.

[0205] Preparation of receptor microspheres with reference material:

[0206] Dissolve 0.1g of carboxylated polystyrene spheres with a particle size of 100nm in 100mL of ultrapure water and sonicate to form a dispersed phase; add 0.5mL to 5mL each of 2wt% sodium dodecylbenzenesulfonate and 1wt% ethylenediamine polyoxyethylene polyoxypropylene block polyether to the dispersion and stir to obtain an aqueous phase;

[0207] The reference substance, buffer, and luminescent agent were dispersed in 10 mL of tetrahydrofuran solution at a molar ratio of 1:10:50. After the solution was prepared, it was quickly added to the aqueous phase above. The temperature was then gradually increased to 50 °C, and the mixture was stirred continuously for 10 h before centrifugation. The obtained carboxylated polystyrene spheres were washed twice with ultrapure water and ethanol and stored in ultrapure water to prepare donor microspheres. These microspheres were then stored at room temperature in the dark for later use.

[0208] Preparation of donor microspheres that do not contain reference materials:

[0209] Dissolve 0.1g of carboxylated polystyrene spheres with a particle size of 100nm in 100mL of ultrapure water and sonicate to form a dispersed phase; add 0.5mL to 5mL each of 2wt% sodium dodecylbenzenesulfonate and 1wt% ethylenediamine polyoxyethylene polyoxypropylene block polyether to the dispersion and stir to obtain an aqueous phase;

[0210] The sensitizer was dispersed in 10 mL of tetrahydrofuran solution. After the solution was prepared, it was quickly added to the aqueous phase. The temperature was then gradually increased to 50 °C and stirred for 10 h. After centrifugation, the obtained carboxylated polystyrene spheres were washed twice with ultrapure water and ethanol and stored in ultrapure water to prepare donor microspheres. The microspheres were then stored at room temperature in the dark for later use.

[0211] The final test results were similar to those of Example 1. This is because the reference substance used in this example is a single molecule, and the emission wavelength of this reference substance molecule meets the requirements for system construction as well as those of the sensitizer, buffer, and luminescent agent. Specifically, the absorption energy level of the reference substance does not overlap with the emission energy levels of the sensitizer, buffer, and luminescent agent, and the wavelengths corresponding to these energy levels differ by more than 10 nm. Regardless of whether the reference substance is introduced into the donor microspheres or the acceptor microspheres, it can function normally and perform the function of reference calibration.

[0212] Similarly, by using the basic schemes of Examples 1 and 2, the reference material can be introduced into both the donor microspheres and the acceptor microspheres simultaneously, and it can also function normally and perform the function of reference calibration.

[0213] Based on the above embodiments, it is demonstrated that the homogeneous detection materials, detection methods, and systems based on reference calibration are feasible and can achieve better results compared to the case without reference calibration. It is worth noting that the significant advantages of the above embodiments confirm the feasibility and advancement of reference calibration in homogeneous detection, reflecting the creativity from academic concept to practical application.

[0214] For example, the reference material can be selected according to the actual situation, and is not limited to the temperature calibration or pseudo-signal calibration shown in this invention. The type of reference material can be selected from other fluorescent materials, upconversion luminescence systems, room temperature phosphorescence systems, organic long afterglow systems, etc., according to the selection criteria and requirements of this invention.

[0215] Moreover, the ways in which the reference material is introduced into the system are diverse, not limited to the coating method shown in this invention. The reference material can be introduced into the system through chemical bonding, charge adsorption, or coating. The chemical bonding method involves covalently linking the reference material to the system (e.g., modifying the reference material with amino groups, which react with the epoxy groups in the system to form chemical bonds). The charge adsorption method involves physically adsorbing the reference material into the system (e.g., the reference material contains a positive charge, which forms an adsorption reaction with the negative charge in the system).

[0216] Regarding wavelength, this invention provides a technical solution greater than 10nm, preferably greater than 30nm, and more preferably greater than 50nm, proving that a wavelength difference greater than 10nm can more effectively avoid signal crosstalk.

[0217] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A long-persistence homogeneous detection material based on reference calibration, comprising a long-persistence detection system, wherein the long-persistence detection system is used to detect analytes, including donor markers and acceptor markers; wherein, Donor markers contain sensitizers; Receptor markers comprise a caching agent and a luminescent agent, characterized in that, It also includes: a reference material, which is used to introduce into the long afterglow detection system to calibrate the detection results of the analyte; The long afterglow detection system does not transfer energy with the reference material; The emission wavelength of the reference material differs from the emission wavelength and / or emission lifetime of the long afterglow detection system. The reference substance includes: a first reference molecule and a second reference molecule; The first reference molecule and the second reference molecule are introduced into the donor marker and the acceptor marker, respectively; Furthermore, the first reference molecule and the second reference molecule constitute a fluorescence resonance energy transfer (FRET) luminescence system.

2. The long-afterglow homogeneous detection material based on reference calibration according to claim 1, characterized in that, The reference substance is introduced into the donor marker and / or the recipient marker.

3. The long-afterglow homogeneous detection material based on reference calibration according to claim 2, characterized in that, The reference material is introduced into the donor and / or acceptor markers through chemical bonding, charge adsorption, or encapsulation.

4. The long-afterglow homogeneous detection material based on reference calibration according to claim 2, characterized in that, When the reference material is introduced into the donor label, the absorption energy level of the reference material does not overlap with the emission energy level of the sensitizer, and the wavelengths corresponding to the energy levels differ by more than 10 nm. When the reference material is introduced into the acceptor label, the absorption energy level of the reference material does not overlap with the emission energy level of the luminescent agent, and the wavelengths corresponding to the energy levels differ by more than 10 nm. When the reference material is introduced into the donor marker and the acceptor marker, the absorption energy level of the reference material does not overlap with the emission energy levels of the sensitizer, the buffer, and the luminescent agent, and the wavelengths corresponding to the energy levels differ by more than 10 nm.

5. The long-afterglow homogeneous detection material based on reference calibration according to claim 2, characterized in that, The reference material is selected from one of the following: fluorescent material, upconversion luminescence system, room temperature phosphorescence system, or organic long afterglow system.

6. The long-afterglow homogeneous detection material based on reference calibration according to claim 5, characterized in that, The reference material is selected from one of the following: cyanine fluorescent dyes, rhodamine fluorescent dyes, fluoroboron dipyrrole fluorescent dyes, rare earth nanomaterials, luminescent complexes, or luminescent polymers.

7. The long-afterglow homogeneous detection material based on reference calibration according to claim 2, characterized in that, The reference material is selected from one of the following: a luminescent temperature probe, a luminescent oxygen probe, or a laser dye.

8. The long-afterglow homogeneous detection material based on reference calibration according to any one of claims 1-7, characterized in that, The sensitizer is selected from dyes or quantum dots; Alternatively, the sensitizer may be selected from quantum dots introduced into the donor label; The buffer and the luminescent agent are linked by chemical bonds; Alternatively, the caching agent and the luminescent agent may be introduced into the receptor marker.

9. The long-afterglow homogeneous detection material based on reference calibration according to any one of claims 1-7, characterized in that, The molar ratio of the sensitizer to the reference substance is 1000:1 to 1:

1.

10. The long-afterglow homogeneous detection material based on reference calibration according to claim 1, characterized in that, If the first reference molecule is an energy donor or acceptor of the FRET luminescent system, then the second reference molecule corresponds to an energy acceptor or donor of the FRET luminescent system. When the first reference molecule is introduced into the donor label, the absorption energy level of the first reference molecule does not overlap with the emission energy level of the sensitizer, and the wavelengths corresponding to the energy levels differ by more than 10 nm. When the second reference molecule is introduced into the receptor marker, the absorption energy level of the second reference molecule does not overlap with the emission energy levels of the buffer and the luminescent agent, and the wavelengths corresponding to the energy levels differ by more than 10 nm.

11. The long-afterglow homogeneous detection material based on reference calibration according to claim 10, characterized in that, Both the first reference molecule and the second reference molecule are selected from one of the following: fluorescent substances, upconversion luminescence systems, room temperature phosphorescence systems, or organic long afterglow systems.

12. The long-afterglow homogeneous detection material based on reference calibration according to any one of claims 1, 10-11, characterized in that, The molar ratio of the first reference molecule to the sensitizer is 1000:1 to 1:1; The molar ratio of the first reference molecule to the second reference molecule is 1:3 to 3:

1.

13. A reference-calibrated long-afterglow homogeneous detection system, applied to the reference-calibrated long-afterglow homogeneous detection material according to any one of claims 1-12 for homogeneous immunoassay, realizing photoexcitation, signal collection, and data analysis and processing of the reference material and the long-afterglow detection system, characterized in that, include: A sample chamber configured to hold a homogeneous immunoreaction system to be tested; A light source device configured to output excitation light that excites a long-afterglow homogeneous detection material containing a reference substance to illuminate the sample chamber; A light acquisition device configured to acquire light from the sample chamber; A detection device configured to receive light output from the acquisition device and generate a spectrum or kinetic curve based on the received light; and A processing device configured for data processing.

14. A method for detecting long-persistent homogeneous phase phases based on reference calibration, providing the long-persistent homogeneous phase phase detection material based on reference calibration as described in any one of claims 1-9, and employing the detection system described in claim 13, characterized in that, Includes the following steps: S1. Provides a standard curve of the optical signal of the parameter to be calibrated and the standard curve of the concentration of the long afterglow light signal of the parameter to be calibrated and the analyte. S2. Provide long afterglow homogeneous detection materials containing reference substances; S3. The sample to be tested, diluent, and long afterglow homogeneous detection material are added to the reaction cup in sequence and stirred evenly to prepare an immunoreaction system. Then the reaction cup is placed in the sample chamber. S4. Turn on the light source device and collect the light signal F1 of the reference material and the light signal F2 of the long afterglow system material by the collection device. S5. The processing device processes the data, substitutes F1 into the standard curve of the parameter to be calibrated - the reference substance light signal, and obtains the value of the parameter to be calibrated. The obtained value of the parameter to be calibrated and F2 are then substituted into the standard curve cluster of the parameter to be calibrated - long afterglow light signal - concentration of the analyte to obtain the concentration of the analyte.

15. The long-persistence homogeneous detection method based on reference calibration according to claim 14, characterized in that, The parameters to be calibrated include environmental factors or instrument factors; Among these factors, environmental factors include temperature or oxygen; instrumental factors include excitation light intensity.

16. A method for detecting long-persistent homogeneous phase phases based on reference calibration, providing the long-persistent homogeneous phase phase detection material based on reference calibration as described in any one of claims 1, 10-12, and employing the detection system described in claim 13, characterized in that, Includes the following steps: S100 provides standard curves for anomaly rate versus reference material optical signal and standard curves for anomaly rate versus long-persistence optical signal. S200 provides a long afterglow signal-analyte concentration standard curve; S300 provides long afterglow homogeneous detection materials containing reference substances; S400. The sample to be tested, diluent, and long afterglow homogeneous detection material are added to the reaction cup in sequence and stirred evenly to prepare an immunoreaction system. Then the reaction cup is placed in the sample chamber. S500, turn on the light source device, and the acquisition device acquires the light signal F1 of the reference material and the light signal F2 of the long afterglow system material; S600: The processing device processes F1 and substitutes it into the standard curve of anomaly rate-reference material light signal to obtain the anomaly rate. The obtained anomaly rate is then substituted into the standard curve of anomaly rate-long afterglow light signal to obtain the long afterglow light signal F3. Finally, F2-F3 are substituted into the standard curve of long afterglow light signal-detection substance concentration to obtain the detection concentration.

17. The long-persistence homogeneous detection method based on reference calibration according to claim 16, characterized in that, The steps for obtaining the standard curves of the anomaly rate versus the reference material optical signal and the anomaly rate versus the long-persistence optical signal are as follows: S1001, The donor marker and acceptor marker containing the reference substance are linked by chemical bonds as component 1, and the long afterglow detection system is used as component 2; S1002. Component 1 and component 2 are allocated according to N different proportions to form N abnormal groups, which are denoted as N different abnormal rates. Component 1 and component 2 of the N abnormal groups are added to the reaction system to be tested, and the detection system described in claim 13 is used for testing to obtain N reference material light signals F01 and N long afterglow light signals F02. The standard curve of the anomalous rate versus the reference material optical signal was obtained by fitting S1003, N F01, and N different anomalous rates; the standard curve of the anomalous rate versus the long-persistence light signal was obtained by fitting N F02 and N different anomalous rates. The steps for obtaining the long afterglow signal-analyte concentration standard curve are as follows: S2001, Provides M different concentrations of the analyte. S2002. Add the long-afterglow homogeneous detection material containing the reference material to the analyte, and test it using the detection system described in claim 13 to obtain M reference material optical signals F011 and M long-afterglow optical signals F022. S2003. Substitute the M reference substance light signals F011 into the standard curve of anomaly rate-reference substance light signal to obtain the anomaly rate. Substitute the obtained anomaly rate into the standard curve of anomaly rate-long afterglow light signal to obtain M long afterglow light signals F033, F022-F033 and M kinds of analyte concentrations to obtain the long afterglow light signal-analyte concentration standard curve. Where N and M are both integers greater than 3.

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