CN116660225A - Long afterglow homogeneous phase detection material, detection method and system based on reference calibration - Google Patents
Long afterglow homogeneous phase detection material, detection method and system based on reference calibration Download PDFInfo
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- CN116660225A CN116660225A CN202310599959.0A CN202310599959A CN116660225A CN 116660225 A CN116660225 A CN 116660225A CN 202310599959 A CN202310599959 A CN 202310599959A CN 116660225 A CN116660225 A CN 116660225A
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Abstract
The invention discloses a long-afterglow homogeneous phase detection material based on reference calibration, which comprises a long-afterglow detection system, wherein the long-afterglow detection system is used for detecting an object to be detected and comprises a donor marker and an acceptor marker, and the donor marker comprises a sensitizer, an acceptor marker buffer agent and a luminescent agent; the material also comprises a reference substance which is used for being introduced into the donor marker and/or the receptor marker and calibrating the detection result of the object to be detected; the long afterglow detection system and the reference substance do not generate energy transfer, and the wavelength difference exists between the luminescence wavelength of the reference substance and the luminescence wavelength of the long afterglow detection system. And discloses a corresponding detection method and a detection system; calibration of environmental and instrument factors can be achieved when a reference substance is introduced into the donor and/or acceptor labels; when the method for detecting the immunity of the long afterglow is calibrated based on the FRET luminous system, the calibration of the non-immunity combination false signal can be realized.
Description
Technical Field
The invention belongs to the field of immunodetection, and relates to a long-afterglow homogeneous detection material based on reference calibration, a detection method and a detection system.
Background
The long afterglow homogeneous detection is one homogeneous detection method for immunological detection, and the method is based on the distance effect of energy transfer between donor microsphere and acceptor microsphere and has long afterglow luminous signal strength to indicate the concentration information of the matter to be detected. Related reagents and methods are disclosed in the CN202010347677.8 patent, wherein the donor microsphere is coated with sensitizer molecules, the acceptor microsphere is coated with a buffer agent and a luminescent agent, and the acceptor microsphere approaches the donor microsphere by means of immune reaction binding and the like. The light source selectively excites the donor microspheres, which then activate the acceptor microspheres by an energy transfer process, ultimately emitting a long afterglow luminescent signal that is detected by an optical detector and used for result analysis. The energy transfer efficiency is inversely related to the distance and falls within the effective range over a distance scale of about 100 nm. The existence of the object to be detected in the sample to be detected and the concentration information thereof can be judged through the intensity detection of the long afterglow luminous signals. The method effectively avoids complex steps such as elution and separation, and can complete a series of steps from incubation to detection in a short time by matching with the high-affinity antibody, so that high-sensitivity detection is realized, and meanwhile, the detection efficiency and cost performance are greatly improved. In addition, long afterglow luminescence is used as a detection signal, so that interference such as autofluorescence is effectively avoided, and a high signal-to-noise ratio can be obtained.
However, in the current detection method, single luminous intensity information is generally adopted as a basis of immunoassay, and the detection method is easily affected by environmental factors such as external environment, and can not exclude false signals generated by the combination of donor microspheres due to non-immune reaction, so that accurate detection results are difficult to obtain.
Disclosure of Invention
In order to solve the problems, the invention discloses a long afterglow homogeneous material based on reference calibration, a detection method and a detection system. Aims at providing a long-afterglow homogeneous phase detection method by introducing an internal reference system to calibrate a detection result, and at the same time, creating a long-afterglow homogeneous phase detection system based on the method.
One of the purposes of the present invention is to provide a long afterglow homogeneous phase detection material based on the long afterglow luminescent reagent (herein referred to as long afterglow detection system) disclosed in the patent of CN202010347677.8, which comprises a donor label and an acceptor label, wherein the donor label contains a sensitizer, and the acceptor label contains a buffer agent and a luminescent agent;
donor labels generally refer to a photochemical energy donor to which is attached a counterpart that specifically binds to the substance to be detected; a photochemical energy donor, i.e. comprising a sensitizer according to the present invention which under photoexcitation generates singlet oxygen;
Receptor labels generally refer to a photochemical energy receptor to which is attached another counterpart that specifically binds to the substance to be detected; photochemical energy acceptors, i.e. comprising a buffering agent and a luminescent agent as described in the present application, wherein the energy acceptor is capable of reacting with singlet oxygen to produce an afterglow luminescent signal.
In the present application, a photochemical energy donor is a different noun concept than an energy donor of FRET; photochemical energy acceptors are a different term concept than FRET energy acceptors.
More preferably, the photochemical energy donor is selected from donor microspheres formed from a carrier matrix with the nanospheres as a sensitizer, and the photochemical energy acceptor is selected from acceptor microspheres formed from a carrier matrix with the nanospheres as a buffer and a luminescent agent.
The application aims at calibrating the test result of the homogeneous detection of the long afterglow system disclosed by the application and the inventor thereof by introducing a reference substance.
In the following, the principle of long afterglow homogeneous detection is explained by taking donor microspheres and acceptor microspheres as photochemical energy donors and photochemical energy acceptors, respectively, as shown in fig. 1, the detection method is based on the effective energy transfer effect generated by the approach (10 nm-100 nm) between the donor marker and the acceptor marker, so that the effect is utilized to homogeneously detect the object to be detected. In this method, a photoexcitation chemical energy donor (donor microsphere, for example, phthalocyanine molecule is wrapped in the donor microsphere) is generally adopted, and a photochemical energy acceptor, i.e. acceptor microsphere, approaches to the donor microsphere through a specific immunoreaction mode, emits long afterglow luminescence with a certain wavelength through an energy transmission process, and then judges whether a target object to be detected exists in an actual detection sample through intensity detection of a long afterglow luminescence signal.
The invention aims to add a reference substance into a donor marker and/or an acceptor marker, eliminate the interference of environment, instrument and equipment or the interference existing in a detection method, and further improve the detection accuracy of the long afterglow material applied to homogeneous phase immunoassay.
The following is a specific scheme of the invention:
the long-afterglow homogeneous detection material based on the reference calibration comprises a long-afterglow detection system, wherein the long-afterglow detection system is used for detecting an object to be detected and comprises a donor marker and an acceptor marker, the donor marker comprises a sensitizer, and the acceptor marker comprises a buffer agent and a luminescent agent;
the detection system also comprises a reference substance, wherein the reference substance is used for being introduced into a long afterglow detection system, and the detection result of the object to be detected is calibrated;
the long afterglow detection system and the reference substance do not generate energy transfer, and the luminescence wavelength of the reference substance is different from the luminescence wavelength and/or the luminescence service life of the long afterglow detection system.
Preferably, the reference substance is introduced to the donor label and/or the acceptor label.
Preferably, the reference substance is introduced into the donor and/or acceptor labels by chemical bond attachment, charge adsorption or coating.
Preferably, the difference between the luminescence wavelength of the reference substance and the luminescence wavelength of the long afterglow detection system is more than 10nm, and the difference between the luminescence wavelength of the reference substance and the luminescence wavelength of the long afterglow detection system is kept more than 10nm, which is better than improvement of the accuracy of the detection result.
Further preferably, the emission wavelength difference is greater than 20nm, more preferably the emission wavelength difference is greater than 30nm.
In addition, the donor and/or acceptor labels, the reference substance may be coated outside the microsphere or coated/embedded within the microsphere, optionally introduced into the donor and/or acceptor microsphere. The carrier matrix in the donor microsphere and the acceptor microsphere can be carrier microsphere disclosed in CN202010347677.8, and the particle size range of the microsphere is preferably 30 nm-1000 nm, and more preferably 100 nm-500 nm.
The sensitizer, the buffer agent and the luminescent agent can be respectively selected from light absorbing agents, photochemical buffer agents and luminescent agents disclosed in CN 202010347677.8.
Preferably, when the reference substance is introduced into the donor label, the absorption energy level of the reference substance does not overlap with the emission energy level of the sensitizer, the energy levels corresponding to wavelengths differing by more than 10nm, preferably by more than 30nm, more preferably by more than 50nm;
when the reference substance is introduced into the acceptor label, the absorption energy level of the reference substance does not overlap with the emission energy level of the luminescent agent, and the energy levels correspond to wavelengths differing by more than 10nm, preferably by more than 30nm, more preferably by more than 50nm;
When the reference substance is introduced into the donor label and the donor label, the absorption energy level of the reference substance does not overlap with the emission energy levels of the sensitizer, the buffer agent and the luminescent agent, and the energy levels correspond to wavelengths differing by more than 10nm, preferably by more than 30nm, more preferably by more than 50nm.
Preferably, the reference substance is selected from one of fluorescent substances, up-conversion luminescent systems, room temperature phosphorescent systems, organic long afterglow systems.
Preferably, the reference substance can be selected from one of cyanine fluorescent dye, rhodamine fluorescent dye, fluoroborodipyrrole fluorescent dye, rare earth nanomaterial, luminescent complex or luminescent polymer.
Preferably, the reference substance may be selected from one of a luminescent temperature probe (reference report chem.soc.rev.,2013,42,7834-7869.), a luminescent oxygen probe (reference report chem.soc.rev.,2010,39,3102-3114.) or a laser dye (reference report chem.rev.,2007,107,1272-1295.).
More specifically, a luminous temperature probe can be selected, and the test result is calibrated by monitoring the temperature of an immune reaction system;
optionally, a luminescent oxygen probe is used to monitor the oxygen concentration or the air pressure where the immune reaction is located to calibrate the test result;
Optionally, a laser dye is used to monitor the intensity of the excitation light to calibrate the test results.
Preferably, the sensitizer is selected from dyes or quantum dots;
or, the sensitizer is selected from the group consisting of being incorporated into the nanoparticle to form a donor microsphere;
the buffer agent and the luminescent agent are connected through chemical bonds, or the buffer agent and the luminescent agent are introduced into the nanometer microsphere to form the receptor microsphere.
Specifically, the sensitizer selects dye or quantum dot, and is directly connected with protein to prepare a donor marker, which is non-particle; after quantum dots are introduced into the nano microspheres to form donor microspheres, connecting proteins on the donor microspheres as donor markers, wherein the quantum dots are particle; the buffer agent and the luminescent agent are directly connected with the protein after being connected through chemical bonds to prepare a receptor marker, and the receptor marker is non-particle; after the buffer agent and the luminescent agent are introduced into the nano-microspheres to form receptor microspheres, the protein is connected to the receptor microspheres as a receptor marker, and the receptor marker is particle.
Preferably, the molar ratio of sensitizer to reference substance is 1000:1 to 1:1, a step of; the preferred molar ratio is 100:1 to 1:1.
specifically, the present inventors have disclosed in other documents that the molar ratio of sensitizer to reference substance is 1000:1 to 1:1, a step of; the preferred molar ratio is 100:1 to 1:1, more preferably 100:1 to 10: and 1, selecting and optimizing according to experimental requirements.
Preferably, the reference substance comprises: a first reference molecule and a second reference molecule;
the long afterglow homogeneous detection system based on reference calibration can be applied to materials in the scheme for homogeneous immunodetection, and realizes light excitation, signal collection and data analysis processing of a reference substance and the long afterglow detection system, and comprises the following steps:
a sample chamber configured to hold a homogeneous immunoreaction system to be detected;
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;
a collection device configured to collect light from the sample chamber;
a detection device configured to receive light output from the collection device and generate a spectrum or a kinetic profile based on the received light; and
processing means configured for data processing.
Preferably, the long-afterglow homogeneous detection method based on the reference calibration adopts the long-afterglow homogeneous detection material based on the reference calibration in the scheme, and the detection system adopting the scheme comprises the following steps:
S1, providing a standard curve of a parameter to be calibrated-reference substance optical signal, a parameter to be calibrated-long afterglow optical signal-concentration standard curve cluster of a substance to be detected;
s2, providing a long-afterglow homogeneous detection material containing a reference substance;
s3, sequentially adding a sample to be detected, a diluent and a long-afterglow homogeneous phase detection material into a reaction cup, uniformly stirring to prepare an immune reaction system, and then placing the reaction cup into the sample chamber;
s4, starting the light source device, collecting the optical signal F1 of the reference substance by the collecting device, and collecting the optical signal F2 of the long afterglow system material;
s5, processing by a processing device, substituting F1 into a standard curve of the parameter to be calibrated-reference substance optical signal to obtain a parameter value to be calibrated, substituting the obtained parameter value to be calibrated and F2 into a standard curve cluster of the parameter to be calibrated-long afterglow optical signal-object concentration to be detected to obtain the object concentration to be detected.
Preferably, the parameter to be calibrated includes an environmental factor or an instrument factor, wherein the environmental factor includes temperature or oxygen, and when the factor to be calibrated is temperature, a luminous temperature probe can be used; when the factor to be calibrated is oxygen or air pressure, a luminous oxygen probe can be selected; when the factor to be calibrated is the intensity of the excitation light, an excitation dye may be used.
An energy transfer phenomenon is generated between two fluorescent molecules which are very close to each other in the FRET system, the emission energy level of the donor fluorescent molecule overlaps with the absorption energy level of the acceptor fluorescent molecule, and when the distance between the two molecules is within 10nm, a fluorescence resonance energy transfer process occurs. The donor fluorescent molecule exhibits characteristic luminescence of the acceptor fluorescent molecule when the distance from the acceptor fluorescent molecule is less than 10 nm.
Taking a particle type as an example, the particle type,
as shown in fig. 1, when the object to be detected is not bound, ideally, the distance between the donor microsphere and the acceptor microsphere is greater than 100nm, and singlet oxygen is difficult to effectively transfer energy, so that a long afterglow luminescence signal is not generated, while in actual detection (fig. 2), because free diffusion approach or physical adsorption binding of the microsphere, the distance between the donor microsphere and the acceptor microsphere is less than 10nm, even zero-distance contact can be generated, and high-efficiency long afterglow luminescence can also occur, so that a false signal can be generated.
The inventor aims at removing false signals by introducing a reference substance into a long-afterglow homogeneous detection material, so that the detection result is more accurate.
As shown in fig. 3, the principle of calibration is explained: introducing a reference substance into the microsphere, optionally introducing a first reference molecule or a second reference molecule into the donor microsphere, introducing a second reference molecule or a first reference molecule into the acceptor microsphere, wherein the first reference molecule and the second reference molecule can form a FRET system; the luminescence wavelength of the reference substance is different from that of the long afterglow detection system, and can be distinguished according to the wavelength; or the long-afterglow luminous life is utilized, and the time resolution technology is adopted to distinguish the luminous signals of the reference substance and the long-afterglow detection system. Normal immunological binding, the distance between the donor and acceptor microballoons is larger than 10nm, and no luminescence phenomenon of FRET system exists, so that the occurrence of the luminescence phenomenon of FRET is the condition that the abnormal distance exists between the donor and acceptor microballoons. And then carrying out anomaly correction and calibrating the luminous signal/intensity, so that the detection result is more accurate.
The specific scheme is as follows:
the invention also provides a long-afterglow homogeneous detection material based on reference calibration, wherein the reference substance comprises a first reference molecule and a second reference molecule;
the first and second reference molecules are introduced to the donor and acceptor labels, respectively, the first and second reference molecules constituting a fluorescence resonance energy transfer FRET luminescent system.
Wherein when the first reference molecule is a FRET donor molecule, the second reference molecule is a FRET acceptor molecule, and when the first reference molecule is a FRET acceptor molecule, the second reference molecule is a FRET donor molecule.
Preferably, the wavelength difference between the luminescence wavelength of the reference substance and the luminescence wavelength of the long afterglow detection system is greater than 10nm; preferably, the wavelength difference is greater than 20nm, more preferably the wavelength difference is greater than 30nm. The difference of the wavelength longer than 10nm is better than the improvement of the accuracy of the detection result, and the larger the difference of the wavelength is, the more favorable the improvement of the accuracy of the detection result, and the preferable difference is 30nm,50nm and 100nm.
Preferably, when the first reference molecule is an energy donor of a FRET system, the second reference molecule is an energy acceptor of the FRET system; when the second reference molecule is an energy donor of the FRET system, the first reference molecule is an energy acceptor of the FRET system;
When the first reference molecule is introduced to the donor label, the absorption energy level of the first reference molecule does not overlap with the emission energy level of the sensitizer, the energy levels corresponding to wavelengths differing by more than 10nm, preferably by more than 30nm, more preferably by more than 50nm;
when a second reference molecule is introduced to the acceptor label, the absorption energy level of the second reference molecule does not overlap with the emission energy levels of the buffer agent and the luminescent agent, and the energy levels correspond to wavelengths differing by more than 10nm, preferably by more than 30nm, more preferably by more than 50nm.
Preferably, the first reference molecule and the second reference molecule are each selected from one of a fluorescent substance, an up-conversion luminescent system, a room temperature phosphorescent system, and an organic long afterglow system.
When the reference substance and the long afterglow system are distinguished, the luminous signals at the respective wavelengths can be collected according to the different wavelengths. In addition, the residual glow of the long-afterglow luminous system can be utilized to distinguish according to the time resolution technology, and the removal of false signals can be realized.
Preferably, the molar ratio of the first reference molecule to the sensitizer is 1000:1 to 1:1, preferably in a molar ratio of 100:1 to 1:1, a step of;
the molar ratio of the first reference molecule to the second reference molecule is 1: 3-3: 1, preferably in a molar ratio of 1:1.
The material can also use the long afterglow homogeneous detection system based on the reference calibration to realize light excitation, signal collection and data analysis processing of the reference substance and the long afterglow detection system.
The invention also discloses a long afterglow homogeneous phase detection method based on reference calibration, which provides the long afterglow homogeneous phase detection material based on reference calibration, and adopts the detection system, and comprises the following steps:
s100, providing a standard curve of an abnormality rate-reference substance optical signal and a standard curve of an abnormality rate-long afterglow optical signal;
s200, providing a long afterglow optical signal-concentration standard curve of the object to be detected;
s300, providing a long-afterglow homogeneous detection material containing a reference substance;
s400, sequentially adding a sample to be detected, a diluent and a long-afterglow homogeneous phase detection material into a reaction cup, uniformly stirring to prepare an immune reaction system, and then placing the reaction cup into the sample chamber;
s500, turning on a light source device, collecting a light signal F1 of a reference substance by a collecting device, and collecting a light signal F2 of a long afterglow system material;
s600, processing by a processing device, substituting F1 into a standard curve of an abnormal rate-reference substance optical signal to obtain an abnormal rate, substituting the obtained abnormal rate into the standard curve of the abnormal rate-long afterglow optical signal to obtain a long afterglow optical signal F3, and substituting F2-F3 into the standard curve of the long afterglow optical signal-to-be-detected object concentration to obtain the to-be-detected concentration.
Wherein the standard curve of the abnormal rate-reference substance optical signal and the standard curve of the abnormal rate-long afterglow optical signal are obtained as follows,
s1001, connecting donor microspheres and acceptor microspheres containing reference substances through chemical bonds to form a component 1,
the long afterglow detection system is used as a component 2,
s1002, distributing the component 1 and the component 2 according to N different proportions, setting the components as N abnormal groups, and recording the N abnormal groups as N different abnormal rates; respectively adding the components 1 and 2 of N abnormal groups into a reaction system to be detected, and testing by using the detection system to respectively obtain N reference substance optical signals F01 and N long afterglow optical signals F02;
s1003, N F01 and N different abnormal rates are fitted to obtain a standard curve of the abnormal rate-reference substance optical signal; and fitting N F02 and N different anomaly rates to obtain a standard curve of anomaly rate-long afterglow optical signals.
Wherein, the obtaining steps of the long afterglow optical signal-concentration standard curve of the object to be detected are as follows,
s2001, providing M substances to be detected with different concentrations,
s2002, respectively adding the long-afterglow homogeneous detection materials containing the reference substances into the objects to be detected, and testing by using the detection system to obtain M reference substance optical signals F011 and M long-afterglow optical signals F022;
S2003, substituting M reference substance optical signals F011 into the standard curves of the abnormal rate-reference substance optical signals to obtain the abnormal rate, substituting the obtained abnormal rate into the standard curve of the abnormal rate-long afterglow optical signals to obtain M long afterglow optical signals F033, F022-F033 and M concentrations of objects to be detected to obtain the standard curve of the long afterglow optical signals-the concentrations of the objects to be detected.
Wherein N and M are integers greater than 3.
In conclusion, the invention can effectively eliminate the influence of environmental factors such as external environment and the like by introducing the reference substances into the long-afterglow homogeneous phase detection material, calibrate the false signals generated by the non-immunoreaction combination of the donor microspheres, and further obtain the accurate detection result.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described below, it being obvious that the drawings in the following description are only illustrative of the principles of the present invention or embodiments, and that other drawings may be obtained from the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a long afterglow homogeneous detection principle;
Wherein, when the distance between the donor microsphere and the receptor microsphere is more than 100nm without being bound with the object to be detected, the effective energy transfer is difficult, and the long afterglow luminescence is avoided; when the distance between the donor microsphere and the receptor microsphere is 10-100nm during immunological binding of the object to be detected, effective energy transfer and energy transmission can be realized, and long afterglow luminescence can be realized;
FIG. 2 is a schematic diagram of the generation of long persistence homogeneous detection artifacts;
the left graph is high-efficiency short-distance energy transfer caused by free diffusion approach;
the right graph shows ultra-high efficiency zero-distance energy transfer caused by physical adsorption combination;
FIG. 3 is a schematic diagram of the correction of the false signal and the reference calibration of the long persistence homogeneous detection;
wherein, the graph i is free diffusion approach pseudo signal correction;
FIG. ii is a physisorption binding artifact correction;
FIG. 4 is a graph showing the long afterglow luminescence spectrum of the europium complex luminescence agent of example 1;
FIG. 5 is a graph showing the up-conversion luminescence spectrum of the up-conversion luminescent material in example 1;
wherein, is excitation light;
FIG. 6 is a graph showing the temperature-optical signal intensity curve of the up-conversion luminescent material in example 1;
FIG. 7 is a graph showing the long afterglow signal-concentration standard curve of the object to be inspected in example 1;
FIG. 8 is a graph showing the concentration of the analyte and the long afterglow luminescence intensity of example 2 before and after calibration.
Reference numerals: 1. donor microspheres; 2. a receptor microsphere; 3. a first counterpart that specifically binds to the biomarker; 4. a second counterpart that specifically binds to the biomarker; 5. exciting a light source; 6. no long afterglow luminescence; 7. long afterglow luminescence; 8. an object to be detected; 9. a first reference molecule; 10. a second reference molecule.
Detailed Description
Example 1
The long afterglow homogeneous detection material, the system and the homogeneous detection method based on the reference calibration are adopted to test C Reaction Protein (CRP) in a whole blood sample
1.1 materials and apparatus used
Sample to be measured: sampling 25 random samples at a time, wherein the random samples comprise normal human whole blood samples and whole blood samples of inflammatory patients;
long afterglow detection system:
sensitizer: phthalocyanine dye with excitation wavelength of 730nm;
luminescent agent: europium complex, the luminescence wavelength is 615nm; the spectrum is shown in figure 4;
caching agent:
reference substance: up-conversion luminescent material (temperature probe), excitation: 730nm, luminescence: 660nm, the spectrum of which is shown in FIG. 5;
a counterpart that specifically binds to the biomarker CRP: CRP-Ab1 monoclonal antibody, CRP-Ab2 monoclonal antibody;
a carrier matrix: carboxyl polystyrene spheres (nanospheres);
the detection system comprises: comprises a sample chamber, a light source device, a collecting device, a detecting device and a processing device,
The sample chamber is configured to hold a homogeneous immunoreaction system to be detected;
the light source device is configured to output excitation light for exciting the long-afterglow homogeneous detection material containing the reference substance to irradiate the sample chamber;
the collection device is configured to collect light from the sample chamber;
the detection means is configured to receive the light output from the acquisition means and to generate a spectrum or a kinetic profile based on the received light;
the processing means is configured for data processing.
1.2 preparation of donor microspheres introduced with reference substances and coating CRP-Ab1 monoclonal antibodies
Preparation of donor microspheres into which reference substances are introduced:
dissolving 0.1g of carboxyl polystyrene spheres with the particle size of 100nm into 100mL of ultrapure water, and performing ultrasonic treatment to form a disperse phase; adding 0.5-5 mL of sodium dodecyl benzene sulfonate with the weight percent of 2% and 1% of ethylenediamine polyoxyethylene polyoxypropylene block polyether into the dispersion liquid, and stirring to obtain a water phase;
the reference substances and the sensitizer are mixed according to a molar ratio of 1:1 is dispersed in 10mL tetrahydrofuran solution, the solution is added into the water phase rapidly after the preparation, then the temperature is gradually raised to 50 ℃, the stirring is continued for 10 hours, and the centrifugation is carried out, the obtained carboxyl polystyrene spheres are washed twice by ultrapure water and ethanol, and are preserved in the ultrapure water to prepare donor microspheres, and are placed in a dark place at normal temperature for standby.
Coupling the counterpart that specifically binds to the biomarker:
centrifuging the prepared donor microsphere at a high speed, washing with BBS buffer solution for 3 times, and finally fixing the volume to 0.5% of solid content, and performing ultrasonic dispersion uniformly to obtain a dispersion liquid;
mu.L of the above dispersion was added to 500. Mu.L of MES and 10mg of EDC, and reacted at room temperature for 2 hours. After the completion of the reaction, the mixture was washed by centrifugation, reconstituted into 500. Mu.L of PBS buffer, and 0.05mg of CRP-Ab1 monoclonal antibody was added thereto, respectively, and reacted at room temperature for 8 hours. After the reaction, the mixture was washed by centrifugation at 35000 rpm and reconstituted into 500. Mu.L of PBS buffer. 10mg of BSA was added thereto and reacted at room temperature for 8 hours. After the reaction, the donor microsphere coated with the CRP-Ab1 antibody is obtained by centrifugal washing, and is re-dissolved in 1.0mL BBS buffer solution and stored at 4 ℃ for standby.
1.3 preparation of receptor microsphere and coated CRP-Ab2 monoclonal antibody
The acceptor microspheres were prepared according to the method for preparing donor microspheres in 1.2, in which instead of sensitizer and reference substance, luminophore and buffer were dispersed in 10mL of tetrahydrofuran solution.
Coupling CRP-Ab2 monoclonal antibody on the receptor microsphere according to a 1.2 coupling method to obtain a receptor microsphere coated with the CRP-Ab2 antibody, re-dissolving the receptor microsphere into 1.0mL BBS buffer solution, and preserving at 4 ℃ for standby.
1.4 preparation of the kit
Preparing microsphere protective agent, which comprises the following components: 20 μL of 20% (w/v) glucose, 20 μL of 10% (w/v) mannitol, 20 μL of 0.5% (w/v) BSA and 20 μL of 0.01 (v/v) Proclin-300. The donor microsphere coated with CRP-Ab1 antibody prepared by 1.2 and the acceptor microsphere coated with CRP-Ab2 antibody prepared by 1.3 are diluted to have the solid content of 0.01% by using the microsphere protective agent, and then are respectively mixed with 100mM BBS buffer (prepared by 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 and pH 7.0-pH 7.6) according to the volume ratio of 1:1, and then are packaged into a reagent bottle 1 as a reagent 1 and a reagent bottle 2 as a reagent 2, so as to obtain a finished product kit. And (5) placing the finished kit at 2-8 ℃ and keeping the kit away from light.
1.5 detection method
Detection of CRP in whole blood samples
1.51 establishing a Standard Curve for the optical signals of the temperature-reference substance
As shown in fig. 6, the temperature-optical signal intensity curve of the luminescent material is up-converted.
1.52 establishing a temperature gradient-long afterglow optical signal-concentration standard curve of the object to be detected
The temperature of the system is controlled to be kept at 10 ℃,
1) The afterglow biomarker CRP antigen was diluted with a diluent (50 mM PBS buffer, containing 1% (w/v) BSA,250mM sodium chloride, 0.1% (w/v) preservative) to 6 dilutions of CRP antigen at sequentially increasing concentrations of 50mg/L, 100mg/L, 150mg/L, 200mg/L, 300mg/L, 400mg/L, respectively, using the dilutions as 0mg/mL;
2) Sequentially adding 2 mu L of one of the CRP antigen dilutions, 98 mu L of the dilution, 150 mu L of the reagent 1 and 150 mu L of the reagent 2 into the same reaction cup, uniformly stirring to prepare an immune reaction system, and incubating for 5min;
3) Irradiating with 730nm excitation light, and comparing the optical signal value F1 of the substance; after 10ms, collecting a long afterglow optical signal value F2, and obtaining a 10-long afterglow optical signal-to-be-detected object concentration standard curve through fitting;
1.53 controlling the system temperature at 25 ℃ and 40 ℃ respectively, repeating the steps 1) 2) 3) to obtain a plurality of long afterglow light signal values F2 respectively, and fitting to obtain a standard curve cluster of 10 ℃, 25 ℃ and 40 ℃ long afterglow light signals-concentration of the object to be detected, as shown in figure 7.
1.54 detection of the biomarker CRP in the sample to be tested (Whole blood)
1) Sequentially adding 2 mu L of whole blood sample to be tested, 98 mu L of diluent, 150 mu L of reagent 1 and 150 mu L of reagent 2 into the same reaction cup, uniformly stirring to prepare an immune reaction system, and incubating for 5min;
2) Irradiating the reaction cup with 730nm excitation light, collecting a reference substance optical signal value F11, closing the excitation light, and collecting a long afterglow signal value F22 after 10 ms;
3) F11 is substituted into a standard curve of a temperature-reference substance optical signal to obtain a temperature value, the temperature value is substituted into a standard curve cluster of a long afterglow optical signal-object concentration to be detected at 10 ℃ and 25 ℃ to obtain the object concentration to be detected, when the temperature is between the two long afterglow optical signals-object concentration to be detected standard curves, the interpolation method is adopted to obtain the long afterglow optical signal-object concentration to be detected standard curve at the temperature again, and then the object concentration to be detected is calculated.
Typically, the results are calculated by direct substitution into a standard curve (e.g., a standard curve developed at room temperature of 25 ℃) for the measured experimental data without consideration of calibration. However, the temperature during the actual detection may deviate from 25 ℃, resulting in inaccurate results. After passing the reference calibration temperature, the resulting analyte concentration is closer to the concentration reference value measured by the medical institution (as shown in table 1).
TABLE 1
Note that: A/B/C is the detected concentration value A reference before calibration (mg/L) B reference after calibration (mg/L) C concentration reference value (mg/L)
Example 2 removal of non-Immunobound artifacts
2.1 materials and apparatus used
Sample to be measured: 10 random samples were taken at a time, including normal human whole blood samples and inflammatory patient whole blood samples:
long afterglow detection system:
sensitizer: platinum benzoporphyrin complex, excitation wavelength 610nm;
luminescent agent: perylene, luminescence wavelength 450nm;
caching agent:
reference substance:
first reference molecule: absorbing 610nm excitation light and emitting 670nm fluorescence;
a second reference molecule: absorbing 670nm light and emitting 700nm fluorescence;
a counterpart that specifically binds to a biomarker: CRP-Ab1 monoclonal antibody, CRP-Ab2 monoclonal antibody;
A carrier matrix: carboxyl polystyrene spheres (nanospheres);
the detection system comprises: comprises a sample chamber, a light source device, a collecting device, a detecting device and a processing device,
the sample chamber is configured to hold a homogeneous immunoreaction system to be detected, in this example the temperature is controlled to be fixed at 37 ℃;
the light source device is configured to output excitation light for exciting the long-afterglow homogeneous detection material containing the reference substance to irradiate the sample chamber;
the collection device is configured to collect light from the sample chamber;
the detection means is configured to receive the light output from the acquisition means and to generate a spectrum or a kinetic profile based on the received light;
the processing means is configured for data processing.
2.2 preparation of donor microspheres incorporating reference substance first reference molecule X1 and coated CRP-Ab1 monoclonal antibody
The preparation procedure was similar to step 1.2, substituting the reference substance for X1 to obtain X1-introduced donor microspheres and X1-introduced donor microspheres coated with CRP-Ab1 antibody, respectively.
2.3 preparation of receptor microspheres incorporating reference substance second reference molecule X2 and coating CRP-Ab1 monoclonal antibody
The preparation steps are similar to those of the step 1.3, and a reference substance X2 is additionally added to respectively obtain receptor microspheres introduced with X2 and coated with CRP-Ab2 antibodies.
2.4 referring to the method of step 1.4, a donor microsphere introduced with X1 and coated with CRP-Ab1 antibody was prepared as reagent 3, and an acceptor microsphere introduced with X2 and coated with CRP-Ab2 antibody was prepared as reagent 4.
Wherein the molar ratio of X1 to X2 is 1:1.
2.5 preparation of systems with anomaly rates of 1%, 5%, 10%, 15%, 20%, 25%, 30%, respectively:
the donor microsphere containing X1 and the acceptor microsphere containing X2 are connected through chemical bonds (the surfaces of the microspheres are connected with bonding groups, such as carboxyl and hydroxyl), so that the component 1 is called, wherein the two microspheres are used in equal amounts;
the donor microsphere containing X1 and the acceptor microsphere containing X2 are directly mixed and then referred to as component 2, wherein the two microspheres are used in equal amounts. The abnormality rate is defined by the ratio of the microsphere concentration of component 1 to component 2, and the ratio of the concentration of component 1 to the concentration of component 2 is 1, taking 10% abnormality rate as an example: 9.
2.6 detection method
2.61 establishing a standard curve of an abnormality rate-reference substance optical signal and a standard curve of an abnormality rate-long afterglow optical signal
The exception rates were 1%, 5%, 10%, 15%, 20%, 25%, 30% for the system prepared with 2.5 (300. Mu.L) and added to 100. Mu.L dilutions (50 mM PBS buffer, containing 1% BSA,250mM sodium chloride, 0.1% preservative) respectively (with or without antigen).
2) And (3) irradiating with 610nm excitation light to respectively obtain a plurality of reference substance optical signal values F01 and a plurality of long afterglow optical signal values F02, and respectively obtaining a standard curve of an anomaly rate-reference substance optical signal and a standard curve of an anomaly rate-long afterglow optical signal through fitting.
2.62 establishing a standard curve of the long afterglow optical signal and the concentration of the object to be detected
1) CRP antigen was diluted to the following concentrations using a dilution: the concentrations were 50mg/L, 100mg/L, 150mg/L, 200mg/L, 300mg/L, 400mg/L, respectively, and the dilutions were used as 0mg/mL.
2) Sequentially adding 2 mu L of one of the CRP antigen dilutions, 98 mu L of the dilution, 150 mu L of the reagent 3 and 150 mu L of the reagent 4 (the molar ratio of the first reference molecule to the second reference molecule is 1:1) into the same reaction cup, uniformly stirring to prepare an immune reaction system, and incubating for 5min;
3) Illuminating with 610nm excitation light to respectively obtain a plurality of reference substance optical signal values F011; after the excitation light is turned off for 10ms, a plurality of long afterglow optical signal values F022 are respectively obtained, the long afterglow optical signal values are obtained through fitting, a plurality of reference substance optical signals F011 are substituted into an abnormal rate-reference substance optical signal standard curve to obtain an abnormal rate, the obtained abnormal rate is substituted into the abnormal rate-long afterglow optical signal standard curve to obtain a plurality of long afterglow optical signals F033, and a plurality of (F022-F033) values and a plurality of CRP concentration values are fitted to obtain a long afterglow optical signal-object concentration standard curve.
2.63 detection of the biomarker CRP in the sample to be tested (Whole blood)
1) Sequentially adding 2 mu L of whole blood sample to be tested, 98 mu L of diluent, 150 mu L of reagent 3 and 150 mu L of reagent 4 into the same reaction cup, uniformly stirring to prepare an immune reaction system, and incubating for 5min;
2) Irradiating the reaction cup with 610nm excitation light, collecting a reference substance optical signal value F011 ', closing the excitation light, and collecting a long afterglow signal value F022', after 10 ms;
3) Substituting F011 'into the standard curve of the abnormal rate-reference substance optical signal to obtain the abnormal rate, substituting the obtained abnormal rate into the standard curve of the abnormal rate-long afterglow optical signal to obtain afterglow signal F033', and substituting (F022 '-F033') into the standard curve of the long afterglow optical signal-to-be-detected object concentration to obtain the concentration of the to-be-detected object.
Typically, the results are calculated by direct substitution into an uncalibrated standard curve for the measured experimental data without consideration of calibration. However, conditions in the actual detection process may have situation differences (e.g., abnormal conditions caused by non-immunological binding differ), resulting in inaccurate results. As shown in fig. 8, the pseudo signal can be well corrected by this method, as is clear from the graphs of the plurality of long afterglow signal values F022", and the plurality of corrected values (F022" -F033 ") and gradient concentrations. After the abnormality rate is calibrated by the reference, the obtained concentration of the object to be detected is closer to the concentration reference value measured by the medical institution (as shown in table 2).
TABLE 2
Note that: A/B/C is the detected concentration value A reference before calibration (mg/L) B reference after calibration (mg/L) C concentration reference value (mg/L)
Example 3
Example 1 was repeated except that the reference substance was introduced into the receptor microsphere.
Preparation of acceptor microspheres into which reference substances are introduced:
dissolving 0.1g of carboxyl polystyrene spheres with the particle size of 100nm into 100mL of ultrapure water, and performing ultrasonic treatment to form a disperse phase; adding 0.5-5 mL of sodium dodecyl benzene sulfonate with the weight percent of 2% and 1% of ethylenediamine polyoxyethylene polyoxypropylene block polyether into the dispersion liquid, and stirring to obtain a water phase;
the reference substance, the buffer agent and the luminescent agent are mixed according to the mole ratio of 1:10:50 is dispersed in 10mL tetrahydrofuran solution, the solution is added into the water phase rapidly after the preparation, then the temperature is gradually raised to 50 ℃, the stirring is continued for 10 hours, and the centrifugation is carried out, the obtained carboxyl polystyrene spheres are washed twice by ultrapure water and ethanol, and are preserved in the ultrapure water to prepare donor microspheres, and are placed in a dark place at normal temperature for standby.
Preparation of donor microspheres without reference substance accordingly:
dissolving 0.1g of carboxyl polystyrene spheres with the particle size of 100nm into 100mL of ultrapure water, and performing ultrasonic treatment to form a disperse phase; adding 0.5-5 mL of sodium dodecyl benzene sulfonate with the weight percent of 2% and 1% of ethylenediamine polyoxyethylene polyoxypropylene block polyether into the dispersion liquid, and stirring to obtain a water phase;
Dispersing sensitizer in 10mL tetrahydrofuran solution, rapidly adding into the water phase after the solution preparation is completed, gradually heating to 50 ℃, continuously stirring for 10 hours, centrifuging, washing the obtained carboxyl polystyrene spheres twice with ultrapure water and ethanol, preserving in the ultrapure water to prepare donor microspheres, and keeping the donor microspheres at normal temperature in a dark place for standby.
The final test results were similar to those of example 1. Because the reference substance used in this embodiment is a single molecule, and the emission wavelength of the reference substance molecule, the sensitizer, the buffer agent, and the luminescent agent all meet the requirements of system construction, that is, the absorption energy level of the reference substance and the emission energy levels of the sensitizer, the buffer agent, and the luminescent agent do not overlap, and the difference of the wavelengths corresponding to the energy levels is greater than 10nm. The reference substance can be introduced into the donor microsphere or the acceptor microsphere, and can work normally to perform the function of reference calibration.
Similarly, the basic protocols of examples 1 and 2 are used to introduce the reference substance into both donor and acceptor microspheres at the same time, and to perform the function of reference calibration.
According to the above examples, homogeneous detection materials, detection methods and systems based on reference calibration have proven to be practical and to achieve superior results compared to the case without reference calibration. It is worth noting that the significant advantages of the above examples demonstrate the feasibility and advances that reference calibration has in homogeneous assays, reflecting the creative from academic ideas to practical applications.
For example, the reference substance may be selected according to the actual situation, and not only includes the temperature calibration or the pseudo signal calibration shown in the present invention. The type of reference substance can be selected from other fluorescent substances, up-conversion luminescent systems, room temperature phosphorescent systems, organic long afterglow systems, etc. according to the selection criteria and requirements of the present invention.
Moreover, the manner in which the reference substance is introduced into the system is also varied and does not merely comprise the coating method presented in the present invention. The reference substance can be introduced into the system by chemical bonding, charge adsorption and coating, wherein the chemical bonding method is to covalently bond the reference substance to the system (for example, the reference substance modifies amino group to react with epoxy groups contained in the system to form chemical bond coupling), and the charge adsorption method is to physically adsorb the reference substance to the system (for example, the reference substance contains positive charges and forms adsorption with negative charges contained in the system).
Regarding the wavelength, the invention provides a technical scheme of more than 10nm, preferably more than 30nm and more preferably more than 50nm, and the signal crosstalk can be effectively avoided after the wavelength difference is more than 10 nm.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art. Thus, the present invention is not intended 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 (18)
1. A long afterglow homogeneous detection material based on a reference calibration, comprising a long afterglow detection system for detecting an object to be detected, comprising a donor marker and an acceptor marker; wherein the donor label comprises a sensitizer; the receptor marker comprises a buffer agent and a luminescent agent, and is characterized in that,
further comprises: the reference substance is used for being introduced into a long afterglow detection system, and the detection result of the object to be detected is calibrated;
the long afterglow detection system and the reference substance do not generate energy transfer;
the reference substance has a luminescence wavelength different from the luminescence wavelength and/or luminescence lifetime of the long afterglow detection system.
2. A long-afterglow homogeneous assay material based on a reference calibration as defined in claim 1, wherein,
the reference substance is introduced into the donor label and/or the acceptor label.
3. A long-afterglow homogeneous assay material based on a reference calibration as defined in claim 2, characterized in that,
the reference substance is introduced into the donor and/or acceptor labels by chemical bonding, charge adsorption or coating.
4. A long-afterglow homogeneous assay material based on a reference calibration as defined in claim 2, characterized in that,
When the reference substance is introduced into the donor label, the absorption energy level of the reference substance is not overlapped with the emission energy level of the sensitizer, and the wavelengths corresponding to the energy levels are different by more than 10nm;
when the reference substance is introduced into the receptor marker, the absorption energy level of the reference substance is not overlapped with the emission energy level of the luminescent agent, and the wavelength corresponding to the energy level is different by more than 10nm;
when the reference substance is introduced into the donor label and the acceptor label, the absorption energy level of the reference substance is not overlapped with the emission energy levels of the sensitizer, the buffer agent and the luminescent agent, and the energy levels correspond to wavelengths which differ by more than 10nm.
5. A long-afterglow homogeneous assay material based on a reference calibration as defined in claim 2, characterized in that,
the reference substance is selected from one of fluorescent substance, up-conversion luminescence system, room temperature phosphorescence system or organic long afterglow system.
6. A long-afterglow homogeneous assay material based on a reference calibration as defined in claim 5, wherein,
the reference substance is selected from one of cyanine fluorescent dye, rhodamine fluorescent dye, fluoroborodipyrrole fluorescent dye, rare earth nano material, luminescent complex or luminescent polymer.
7. A long-afterglow homogeneous assay material based on a reference calibration as defined in claim 2, characterized in that,
the reference substance is selected from one of a luminescent temperature probe, a luminescent oxygen probe or a laser dye.
8. A long-afterglow homogeneous detection material based on a reference calibration according to claim 1 to 7, characterized in that,
the sensitizer is selected from dye or quantum dot;
or, the sensitizer is selected from quantum dots into which a donor label is introduced;
the buffer agent and the luminescent agent are connected through a chemical bond;
alternatively, the buffer and the luminescent agent are incorporated into a receptor label.
9. A long-afterglow homogeneous detection material based on a reference calibration according to claim 1 to 7, characterized in that,
the molar ratio of the sensitizer to the reference substance is 1000:1 to 1:1.
10. a long-afterglow homogeneous assay material based on a reference calibration as defined in claim 1, wherein,
the reference substance comprises: a first reference molecule and a second reference molecule;
the first and second reference molecules are introduced to the donor and acceptor labels, respectively;
and the first reference molecule and the second reference molecule constitute a fluorescence resonance energy transfer FRET luminescent system.
11. A long-afterglow homogeneous assay material based on a reference calibration as defined in claim 10, characterized in that,
the first reference molecule is an energy donor or acceptor of a FRET light-emitting system, and the second reference molecule is an energy acceptor or donor of the FRET light-emitting system;
when the first reference molecule is introduced into the donor label, the absorption energy level of the first reference molecule is not overlapped with the emission energy level of the sensitizer, and the energy level corresponds to a wavelength difference of more than 10nm;
when the second reference molecule is introduced into the receptor marker, the absorption energy level of the second reference molecule is not overlapped with the emission energy levels of the buffer agent and the luminescent agent, and the wavelength difference corresponding to the energy levels is more than 10nm.
12. A long-afterglow homogeneous assay material based on a reference calibration as defined in claim 11,
the first reference molecule and the second reference molecule are both selected from one of fluorescent substances, up-conversion luminescence systems, room temperature phosphorescence systems or organic long afterglow systems.
13. A long-afterglow homogeneous detection material based on a reference calibration according to claim 10 to 12, characterized in that,
the molar ratio of the first reference molecule to the sensitizer is 1000:1 to 1:1, a step of;
The molar ratio of the first reference molecule to the second reference molecule is 1: 3-3: 1.
14. the long afterglow homogeneous detection system based on reference calibration realizes light excitation, signal collection and data analysis processing of the reference substance and the long afterglow detection system,
comprising the following steps:
a sample chamber configured to hold a homogeneous immunoreaction system to be detected;
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;
a collection device configured to collect light from the sample chamber;
a detection device configured to receive light output from the collection device and generate a spectrum or a kinetic profile based on the received light; and
processing means configured for data processing.
15. A method for long persistence homogeneous detection based on a reference calibration, providing a long persistence homogeneous detection material based on a reference calibration as defined in any one of claims 1-9, employing the detection system of claim 14, comprising the steps of:
s1, providing a standard curve of a parameter to be calibrated-reference substance optical signal, a parameter to be calibrated-long afterglow optical signal-concentration standard curve cluster of a substance to be detected;
S2, providing a long-afterglow homogeneous detection material containing a reference substance;
s3, sequentially adding a sample to be detected, a diluent and a long-afterglow homogeneous phase detection material into a reaction cup, uniformly stirring to prepare an immune reaction system, and then placing the reaction cup into the sample chamber;
s4, starting the light source device, collecting the optical signal F1 of the reference substance by the collecting device, and collecting the optical signal F2 of the long afterglow system material;
s5, processing by a processing device, substituting F1 into a standard curve of the parameter to be calibrated-reference substance optical signal to obtain a parameter value to be calibrated, substituting the obtained parameter value to be calibrated and F2 into a standard curve cluster of the parameter to be calibrated-long afterglow optical signal-object concentration to be detected to obtain the object concentration to be detected.
16. The reference calibration-based long persistence homogeneous assay method of claim 15, wherein,
the parameters to be calibrated comprise environmental factors or instrument factors;
wherein the environmental factors include temperature or oxygen; instrument factors include excitation light intensity.
17. A long afterglow homogeneous detection method based on reference calibration, providing a long afterglow homogeneous detection material based on reference calibration as defined in any one of claims 1, 10-13, using the detection system as defined in claim 14, characterized by comprising the steps of:
S100, providing a standard curve of an abnormality rate-reference substance optical signal and a standard curve of an abnormality rate-long afterglow optical signal;
s200, providing a long afterglow optical signal-concentration standard curve of the object to be detected;
s300, providing a long-afterglow homogeneous detection material containing a reference substance;
s400, sequentially adding a sample to be detected, a diluent and a long-afterglow homogeneous phase detection material into a reaction cup, uniformly stirring to prepare an immune reaction system, and then placing the reaction cup into the sample chamber;
s500, turning on a light source device, collecting a light signal F1 of a reference substance by a collecting device, and collecting a light signal F2 of a long afterglow system material;
s600, processing by a processing device, substituting F1 into a standard curve of an abnormal rate-reference substance optical signal to obtain an abnormal rate, substituting the obtained abnormal rate into the standard curve of the abnormal rate-long afterglow optical signal to obtain a long afterglow optical signal F3, and substituting F2-F3 into the standard curve of the long afterglow optical signal-to-be-detected object concentration to obtain the to-be-detected concentration.
18. The reference calibration-based long persistence homogeneous assay method of claim 17, wherein,
the standard curve of the abnormal rate-reference substance optical signal and the standard curve of the abnormal rate-long afterglow optical signal are obtained by the following steps:
S1001, connecting a donor marker and an acceptor marker containing a reference substance through a chemical bond to form a component 1, and taking a long afterglow detection system as a component 2;
s1002, distributing the component 1 and the component 2 according to N different proportions, setting the components as N abnormal groups, and recording the N abnormal groups as N different abnormal rates; respectively adding the components 1 and 2 of N abnormal groups into a reaction system to be detected, and testing by using the detection system of claim 14 to respectively obtain N reference substance optical signals F01 and N long afterglow optical signals F02;
s1003, N F01 and N different abnormal rates are fitted to obtain a standard curve of the abnormal rate-reference substance optical signal; fitting N F02 and N different anomaly rates to obtain a standard curve of anomaly rate-long afterglow optical signals;
the method for obtaining the standard curve of the concentration of the long afterglow optical signal-object to be detected comprises the following steps,
s2001, providing M kinds of objects to be detected with different concentrations,
s2002, respectively adding the long-afterglow homogeneous detection materials containing the reference substances into the objects to be detected, and testing by using the detection system of claim 14 to obtain M reference substance optical signals F011 and M long-afterglow optical signals F022;
s2003, substituting M reference substance optical signals F011 into the standard curves of the abnormal rate-reference substance optical signals to obtain the abnormal rate, substituting the obtained abnormal rate into the standard curve of the abnormal rate-long afterglow optical signals to obtain M long afterglow optical signals F033, (F022-F033) and M concentrations of objects to be detected to obtain the standard curve of the long afterglow optical signals-the concentrations of the objects to be detected;
Wherein N and M are integers greater than 3.
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