CN116429753B

CN116429753B - CEA detection kit and use method thereof

Info

Publication number: CN116429753B
Application number: CN202310110389.4A
Authority: CN
Inventors: 张黎明; 李建武; 康蔡俊; 洪琳; 黄正铭; 李临
Original assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd
Current assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd
Priority date: 2023-02-13
Filing date: 2023-02-13
Publication date: 2024-07-19
Anticipated expiration: 2043-02-13
Also published as: CN116429753A

Abstract

The invention relates to the technical field of photo-excitation chemiluminescence, in particular to a CEA detection kit and a use method thereof. Based on a photosensitive reagent with the photosensitive quantity ps of 1.34< PS <16.28, the invention provides a CEA detection kit, and the detection kit comprises a luminous reagent, a biotin reagent and a photosensitive reagent, wherein the luminous reagent is CEA antibody coated luminous particles, the biotin reagent is biotin-marked CEA antibody, and the photosensitive reagent is streptavidin coated by photosensitive microsphere. The invention also discloses a method for measuring the concentration of carcinoembryonic antigen CEA in a human serum or plasma sample, and a use method thereof, wherein the detection principle of the method is a double-antibody sandwich method, and the method has the advantages of high sensitivity and good precision.

Description

CEA detection kit and use method thereof

Technical Field

The invention relates to the technical field of photo-excitation chemiluminescence, in particular to a CEA detection kit and a use method thereof.

Background

Carcinoembryonic antigen (carcinoembryonic antigen, CEA) is a tumor-associated antigen extracted from colon cancer and embryonic tissue, is an acidic glycoprotein with human embryonic antigen properties, exists on the surface of cancer cells differentiated from endodermal cells, and is a structural protein of cell membranes. Formed in the cytoplasm, secreted out of the cell through the cell membrane and then into the surrounding body fluids. Therefore, the sample can be detected from various body fluids and excretions such as serum, cerebrospinal fluid, milk, gastric juice, hydrothorax and ascites, urine, feces, and the like. Serum CEA is a relatively broad-spectrum tumor marker. Clinically, the method can be used for auxiliary diagnosis of common tumors such as colon cancer, rectal cancer, lung cancer, breast cancer, esophagus cancer, pancreatic cancer, gastric cancer, metastatic liver cancer and the like. Other malignant tumors such as medullary thyroid carcinoma, cholangiocarcinoma, urinary malignant tumors and the like have positive rates with different degrees.

The basic principle of photoexcitation is a homogeneous immune response. It is based on two particles surface coated antigens or antibodies, forming immune complexes in the liquid phase, drawing the two particles closer together. Under the excitation of laser, the transfer of singlet oxygen between particles occurs, and then high-level red light is generated, and the photon number is converted into the concentration of the analyte through a single photon counter and mathematical fitting. When the sample does not contain an analyte, immune complexes cannot be formed between the two particles, the distance between the two particles exceeds the propagation range of singlet oxygen, the singlet oxygen is rapidly quenched in a liquid phase, and no high-energy red light is generated during detection.

The photosensitive reagent is one of indispensable important components in a commodity kit, and has the function that the photosensitive microsphere in the reagent can generate singlet oxygen after being excited by external excitation light, and the singlet oxygen transfers energy to the luminescent microsphere within 200nm from the photosensitive microsphere, so that a chemiluminescent signal can be generated finally, and the detection of an unknown object is realized. The final clinical application and chemiluminescence detection results of the photo-activated chemiluminescence detection kit product are affected by the selection of the concentration of the photosensitive dye filled in the photosensitive microspheres, the efficiency and time of generating singlet oxygen in the liquid phase of the photosensitive microspheres, the production cost of the photosensitive reagent and the like. Currently, the prior art lacks high performance, photosensitive reagents that meet clinical testing requirements.

Therefore, the CEA detection kit and the using method thereof are provided with important practical significance.

Disclosure of Invention

In view of this, the kit provided by the invention comprises CEA reagent 1 (CEA antibody coated luminescent particles), CEA reagent 2 (biotin-labeled CEA antibody) and a photosensitive reagent, and the concentration of carcinoembryonic antigen CEA in human serum or plasma samples is quantitatively detected by adopting a double-antibody sandwich immunofluorescence excitation chemiluminescence method under homogeneous conditions.

In order to achieve the above object, the present invention provides the following technical solutions:

In a first aspect, the present invention provides the use of a photosensitive microsphere in a microsphere composition, a reagent set, a kit, a detection system and/or a detection device for detecting CEA,

Detecting a chemiluminescent signal generated by a luminescent complex formed by the luminescent microsphere-CEA immune complex-photosensitive microsphere;

The luminescent microsphere comprises a carrier and a luminescent substance carried by the carrier, wherein the luminescent substance can react with singlet oxygen to generate chemiluminescence;

The photosensitive microsphere comprises a carrier and a photosensitive substance carried by the carrier, wherein the photosensitive substance can generate singlet oxygen under the excitation of light; the sensitization amount Ps of the sensitization microsphere is between 1.34 and 16.28; the sensitization amount ps=od _λ1/C₂*10³, wherein:

The OD _λ1 is an absorbance value corresponding to a maximum absorption peak of a wavelength-absorbance curve obtained by full-wavelength scanning of the photosensitive microsphere with the concentration of C ₂ in a visible light region ranging from 300nm to 800nm, and the lambda ₁ is a wavelength corresponding to the maximum absorption peak; the unit of C ₂ is the concentration of the photosensitive microsphere in the photo-excitation chemiluminescence detection, and the unit of C ₂ is ug/ml.

In some embodiments of the present invention,

Concentration of the photosensitive microsphere

Where k is the corresponding slope in the linear relationship of carrier concentration-absorbance curve and b is the corresponding intercept in the linear relationship of the carrier concentration-absorbance curve; OD _λ2 is the corresponding absorbance value of the photosensitive microsphere at wavelength λ ₂; the carrier concentration-absorbance curve is a curve obtained by adopting a plurality of carriers with different concentrations at a wavelength lambda ₂; the wavelength lambda ₂ is a wavelength corresponding to the absorbance value of the photosensitive microsphere with the same concentration and the carrier with the same or similar absorbance value in the wavelength-absorbance curve.

In some embodiments of the invention, the C ₂ is selected from 10ug/ml to 200ug/ml.

In some embodiments of the invention, the linear relationship of carrier concentration-absorbance curve is y=kx+b, wherein:

x is different concentrations of the carrier with preset particle size, y is absorbance value of the carrier at the corresponding concentration, k is slope, and b is intercept.

In some embodiments of the invention, the wavelength λ ₂ is selected from any wavelength value for which the ratio of OD _{Photosensitive microsphere}/OD_{carrier body} is within 0.85 to 1.15, and the wavelength λ ₂ is not equal to the wavelength λ ₁;

Wherein, OD _{Photosensitive microsphere} and OD _{carrier body} are absorbance values corresponding to the same wavelength value of the photosensitive microsphere and the carrier with the same concentration in the range of 300nm to 800nm respectively.

In some embodiments of the invention, the wavelength lambda ₂ is 400nm to 600nm.

In some embodiments of the present invention, the photosensitive microsphere is a carrier filled with a photosensitive material, wherein the wavelength lambda ₁ is a wavelength corresponding to a maximum absorption peak in a wavelength-absorbance curve obtained by full-wavelength scanning of the photosensitive material in a visible light region of 300nm to 800 nm.

In some embodiments of the invention, the wavelength lambda ₁ is 600nm to 700nm.

In some embodiments of the invention, the photosensitive microsphere is prepared according to a mass ratio of the carrier to the photosensitive substance of 10 (0.04-4).

In some embodiments of the invention, the carrier has a particle size of 190nm to 280nm.

In some embodiments of the invention, the surface of the photosensitive microsphere is not coated with a polysaccharide, and the surface of the photosensitive microsphere is connected with an avidin, wherein the avidin is selected from the group consisting of ovalbumin, egg yolk avidin, streptavidin, neutravidin and avidin-like molecules, preferably streptavidin.

In some embodiments of the invention, the photosensitive microsphere has a photosensitivity Ps of between 4.07 and 16.28.

In a second aspect, the invention also provides microsphere compositions for detecting CEA, comprising luminescent microspheres and the photoactive microspheres.

In a third aspect, the present invention also provides a reagent combination for detecting CEA, comprising a luminescent reagent and a photoactive reagent;

the photosensitive agent comprises a buffer solution and the photosensitive microspheres stored in the buffer solution;

the luminescent reagent comprises luminescent microspheres and substances capable of reacting with CEA.

In some embodiments of the invention, the sugar content in the buffer solution is 1 g.+ -. 0.2g per liter of volume.

In some embodiments of the present invention, the reagent combination further comprises a labeling reagent comprising a label and the substance capable of reacting with CEA;

The photosensitizing agent also includes a substance capable of binding to the label;

Preferably, the substance capable of reacting with CEA includes, but is not limited to: CEA antibodies;

preferably, the markers include, but are not limited to: biotin;

Preferably, the substance capable of binding to the label includes, but is not limited to: streptavidin.

In a fourth aspect, the invention also provides a kit for detecting CEA, comprising one or more of any of the following, and a substance capable of reacting with an analyte, acceptable adjuvant, adjuvant or carrier;

(I) The photosensitive microsphere; or (b)

(II) the microsphere composition; or (b)

(III) the reagent combination;

such carriers include, but are not limited to: reagent bottles, reagent cards, test strips or chips.

In some embodiments of the invention, the kit comprises a luminescent reagent, a labeling reagent, and a photosensitizing reagent;

the luminescent agents include, but are not limited to: CEA antibody coated luminescent microparticles;

such labeling agents include, but are not limited to: a biotin-labeled CEA antibody;

The photosensitizing agent includes, but is not limited to: streptavidin coated photosensitive microsphere.

In a fifth aspect, the present invention also provides a system or apparatus for detecting CEA, comprising one or more of any of the following, and an acceptable module or component;

(I) The photosensitive microsphere; or (b)

(II) the microsphere composition; or (b)

(III) the reagent combination; or (b)

(IV) the kit.

The beneficial effects of the invention include, but are not limited to:

According to the technical scheme, after the values of the absorbance value OD _λ1 and the concentration C ₂ are determined, the light sensing quantity Ps of the light sensing microsphere can be determined according to the ratio of the absorbance value OD _λ1 to the concentration C ₂. When the value of the sensitization quantity Ps of the sensitization microsphere is between 1.34 and 16.28, the sensitization microsphere is applied to the sensitization reagent to carry out a mixed reaction with the luminous microsphere, so that the intensity of a chemiluminescent signal of the luminous microsphere can meet the requirements in photoexcitation chemiluminescence detection, the fluctuation of a detection result caused by the influence of other interference factors on the chemiluminescent signal is reduced, the detection result has consistency and repeatability in clinical application, and the detection result has more definite distinction and higher precision. The photosensitive microsphere provided by the application has the advantages that the performance standard for execution in the application of the photosensitive microsphere to the photo-excitation chemical detection is given through definite numerical limitation, the operability is definite, and the photosensitive microsphere is suitable for popularization of industry specifications.

Based on a photosensitive reagent with the photosensitive quantity ps of 1.34< PS <16.28, the CEA detection kit provided by the invention comprises a luminous reagent, a biotin reagent and a photosensitive reagent, wherein the luminous reagent is CEA antibody coated luminous particles, the biotin reagent is biotin-labeled CEA antibody, and the photosensitive reagent is streptavidin coated by photosensitive microsphere, and the kit has better performance.

The invention discloses a method for measuring the concentration of carcinoembryonic antigen CEA in a human serum or plasma sample, and simultaneously discloses a use method, wherein the detection principle of the method is a double-antibody sandwich method, and the method has the advantages of high sensitivity and good precision.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a graph showing the particle size results of a 20ug/ml carrier measured by a particle sizer according to an embodiment of the present application;

FIG. 2 is a graph showing the particle size results of 20ug/ml photosensitive microspheres measured by a particle size meter according to an embodiment of the present application;

FIG. 3 shows a wavelength-absorbance curve of a photosensitive material according to an embodiment of the application;

FIG. 4 shows wavelength-absorbance curves for different concentrations of carrier according to embodiments of the application;

FIG. 5 shows wavelength-absorbance curves for various concentrations of photosensitive microspheres according to an embodiment of the application;

FIG. 6 shows a wavelength-absorbance curve of 10 μg/ml of carrier and photosensitive microspheres according to an embodiment of the application;

FIG. 7 shows a carrier concentration-absorbance curve of a carrier at a wavelength of 500nm according to an embodiment of the application;

FIG. 8 shows a concentration-amount of photosensitive material curve for a specific embodiment of the present application;

FIG. 9 shows a method of using the kit of example 5.

Detailed Description

The invention discloses a CEA detection kit and a use method thereof, and a person skilled in the art can refer to the content of the CEA detection kit and properly improve the implementation of technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.

It should be understood that although the terms "first," "second," "third," etc. may be used herein to describe various information, these information should not be limited by these terms. These terms are only used to distinguish one type of information from another. For example, first information may also be referred to as second information, and similarly, second information may also be referred to as first information, without departing from the scope of the application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.

The luminescent microspheres may be polymer particles filled with a luminescent compound formed by coating a functional group on a first support, and may be referred to as luminescent microspheres or luminescent particles. The surface of the luminous microsphere is provided with hydrophilic carboxyl glucan, and the interior of the luminous microsphere is provided with a chemical hair composition capable of reacting with active oxygen (such as singlet oxygen). In some embodiments of the invention, the chemical composition undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate, which may decompose while or subsequently emit light. In some preferred embodiments of the present invention, the luminescent composition, such as europium complexes, is merely illustrative and not limiting.

In the present application, the photosensitive microsphere includes a second carrier and a photosensitive substance carried by the carrier. The second carrier (also referred to as blank microspheres) may be polymer particles, and the photosensitive material may be coated on the surface of the carrier and/or filled in the second carrier. The photosensitive material may be capable of generating active oxygen (e.g., singlet oxygen) under light excitation, and the polymer particles may be polystyrene microspheres, or may be microspheres of other materials for detection, which is not limited herein. The photosensitive substance may be, for example, a photosensitizer or a photosensitive dye, which may be a photosensitive substance known in the art, such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll, and is not limited thereto. The photosensitive microspheres may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Examples of other sensitizers include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and singlet oxygen is released by heating these compounds or by direct absorption of light by these compounds.

The immune complex refers to a complex obtained by combining an antibody with an antigen, and the immune complex is an antigen-antibody, an antigen-antibody-antigen, an antibody-antigen-antibody, an antigen-antibody-secondary antibody and the like. The luminous compound refers to a compound formed by directly or indirectly connecting luminous microspheres and photosensitive microspheres, and can receive laser irradiation with specific wavelength to generate chemical reaction to emit detectable light.

In one embodiment, the photosensitive microsphere for photoexcitation chemiluminescence detection has a photosensitive amount Ps between 1.34 and 16.28; the light sensing amount Ps of the photosensitive microsphere is determined according to the following formula (1).

Ps＝ OD_λ1 /C₂*10³ (1)

Wherein OD _λ1 is an absorbance value corresponding to a maximum absorption peak of a wavelength-absorbance curve obtained by full-wavelength scanning of a photosensitive microsphere with a concentration of C ₂ in a visible light region ranging from 300nm to 800nm, and lambda ₁ is a wavelength corresponding to the maximum absorption peak; c ₂ is the concentration of the photosensitive microsphere during photo-induced chemiluminescence detection, and C ₂ unit is ug/ml.

In the present application, the photosensitive microsphere includes a carrier and a photosensitive substance carried by the carrier. The carrier can be polymer particles, and the photosensitive substance can be coated on the surface of the carrier and/or filled in the carrier. The photosensitive material may be capable of generating active oxygen (e.g., singlet oxygen) under light excitation, and the polymer particles may be polystyrene microspheres, or may be microspheres of other materials for detection, which is not limited herein. The photosensitive substance may be, for example, a photosensitizer or a photosensitive dye, which may be a photosensitive substance known in the art, such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll, and is not limited thereto. The photosensitive microspheres may also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Examples of other sensitizers include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and singlet oxygen is released by heating these compounds or by direct absorption of light by these compounds.

Further, the photosensitive microsphere with the concentration of C ₂ dissolved in the buffer solution is subjected to full-wavelength scanning by using visible light in the range of 300-800 nm through a spectrophotometer and other devices, absorbance values corresponding to different wavelength scanning are read, a corresponding wavelength-absorbance curve is generated, and after the wavelength lambda ₁ is selected from the wavelength corresponding to the maximum characteristic peak (namely the maximum absorption peak) of the photosensitive microsphere in the wavelength-absorbance curve; Correspondingly, the absorbance value corresponding to the maximum characteristic peak in the wavelength-absorbance curve is selected, so that the absorbance value OD _λ1 of the photosensitive microsphere with the concentration of C ₂ at the wavelength lambda ₁ can be determined. Further, in order to ensure accuracy of the value of the wavelength λ ₁, in an embodiment, wavelength-absorbance curves of the photosensitive microspheres with different known concentrations and the same photosensitive substance may be measured in advance, and a wavelength corresponding to the maximum characteristic peak may be selected from the wavelength-absorbance curves of the photosensitive microspheres with the respective concentrations as the value of λ ₁. Experiments in the following prove that the maximum characteristic peaks corresponding to the photosensitive microspheres with the same photosensitive substance under different concentrations are the same, and the maximum characteristic peaks of the photosensitive microspheres are the same as the maximum characteristic peaks of the photosensitive substance carried by the photosensitive microspheres, for example, the photosensitive substance is taken as copper phthalocyanine, and the wavelength lambda ₁ of the photosensitive substance and the photosensitive microspheres is 680nm. Therefore, by selecting the wavelength λ ₁ corresponding to the maximum characteristic peak of the wavelength-absorbance curve of the photosensitive microsphere and then determining the corresponding absorbance value OD _λ1, the accuracy of the calculation result of the amount of photosensitive light can be ensured. When the photosensitive microspheres adopt photosensitive substances with different materials, the visible light region in the range of 300 nm-800 nm can be adaptively reused for scanning so as to determine the specific value of the wavelength lambda ₁.

Before use, the initial state of the photosensitive microspheres during storage is generally a lyophilized solid substance or a refrigerated liquid. When the photosensitive microsphere is a solid substance, a buffer solution is required to be added for re-dissolution, and the concentration of the re-dissolved photosensitive microsphere solution is the initial concentration C ₁. When the photosensitive microsphere is stored as a liquid, the concentration at this time is the initial concentration C ₁. When the photo-excitation chemiluminescence detection is carried out, the photosensitive microsphere can directly participate in the detection by adopting the initial concentration, namely the concentration C ₂ of the photosensitive microsphere during the detection at the moment is equal to C ₁; Or the photosensitive microsphere with the initial concentration C ₁ can be diluted and then participates in detection, namely the concentration C ₂ of the photosensitive microsphere during the detection is not equal to the value of C ₁,C₂, namely the true concentration of the photosensitive microsphere after the initial concentration is diluted. It will be appreciated that when the concentration C ₂ of the photosensitive microsphere is changed after the specific value of the wavelength λ ₁ is determined, the corresponding absorbance value OD _λ1 may be correspondingly different, and the value of OD _λ1 is determined according to the actual measurement. After the values of absorbance value OD _λ1 and concentration C ₂ are determined, based on the ratio of absorbance value OD _λ1 to concentration C ₂, the amount of sensitization Ps of the photosensitive microsphere can be determined. When the value of the sensitization quantity Ps of the sensitization microsphere is between 1.34 and 16.28, the sensitization microsphere is applied to the sensitization reagent to react with the luminous microsphere, so that the intensity of a chemiluminescent signal of the luminous microsphere can meet the requirement in photoexcitation chemiluminescence detection, the fluctuation of a detection result caused by the influence of other interference factors on the chemiluminescent signal is reduced, the detection result has consistency and repeatability in clinical application, and the detection result has more definite distinction and higher precision. The photosensitive microsphere provided by the application has the advantages that the performance standard for execution in the application of the photosensitive microsphere to the photo-excitation chemical detection is given through the definite numerical limitation of the photosensitive quantity, the operability is definite, and the photosensitive microsphere is suitable for popularization of industry specifications.

Further, in order to facilitate the definition of the adjustment range of the concentration C ₂ of the photosensitive microsphere, the concentration C ₂ of the photosensitive microsphere is determined according to the following formula (2).

Wherein k is a corresponding slope in a linear relation of a carrier concentration-absorbance curve, b is an intercept in the linear relation of the carrier concentration-absorbance curve, OD _λ2 is an absorbance value corresponding to the photosensitive microsphere at a wavelength lambda ₂, and the carrier concentration-absorbance curve is a curve obtained at a wavelength lambda ₂ by adopting carriers with different concentrations; wavelength lambda ₂ is the wavelength corresponding to the absorbance value of the same concentration of photosensitive microsphere and carrier in the wavelength-absorbance curve.

Specifically, in order to obtain the concentration C ₂ of the photosensitive microsphere satisfying the range of the photosensitive amount Ps, experiments may be performed using the same material and particle size carrier as the photosensitive microsphere to determine the range of the concentration C ₂ of the photosensitive microsphere. It should be noted that, due to the limitation of the manufacturing process of the photosensitive microspheres and the carrier, the definition of "the same particle size", and the like in the present application means that the difference in particle size between the microspheres is ±5nm, and such a small difference in particle size between the microspheres can be regarded as the same particle size. Wherein, a plurality of carriers with different concentrations and the same particle size can be prepared in advance, and the particle size of the carrier is selected from 190 nm-280 nm. And the absorbance values corresponding to the carriers of each concentration are respectively scanned and measured by adopting visible light with the same wavelength lambda ₂, so that a relation curve of the carrier concentration and the absorbance can be established, and then a linear relation corresponding to the carrier concentration and the absorbance is obtained, and the linear relation can be expressed by adopting the following formula (3).

y＝kx+b (3)

Wherein x is different concentrations of carriers with preset particle sizes, y is an absorbance value of the carriers at the corresponding concentration, k is a slope in the formula (2), and b is an intercept in the formula (2). That is, by the correlation calculation of the formula (3), the values of k and b in the formula (2) can be determined, and thus the concentration C ₂ of the photosensitive microsphere can be determined. Further, to determine the values of k and b, in one embodiment, wavelength λ ₂ is selected from any wavelength value for which the ratio of OD _{Photosensitive microsphere}/OD_{carrier body} is within 0.85 to 1.15, and wavelength λ ₂ is not equal to wavelength λ ₁; Wherein, OD _{Photosensitive microsphere} and OD _{carrier body} are absorbance values corresponding to the same wavelength value of the photosensitive microsphere and the carrier with the same concentration in the range of 300nm to 800nm respectively. in this embodiment, the wavelength λ ₂ is not equal to the wavelength λ ₁, that is, the wavelength λ ₂ is a wavelength corresponding to a non-characteristic peak in the wavelength-absorbance curve, that is, a wavelength avoiding a characteristic peak of the photosensitive substance, so as to reduce the influence of the absorbance value of the photosensitive substance on the absorbance value of the carrier. It is understood that the photosensitive microsphere and the carrier having the same microsphere concentration are scanned at the same wavelength in the range of 300nm to 800nm, respectively, and the absorbance value OD _{Photosensitive microsphere} of the photosensitive microsphere at the concentration in the full wavelength range and the absorbance value OD _{carrier body} of the carrier at the concentration in the full wavelength range can be obtained. The applicant researches show that specific experimental data can be checked in the following related content, and the wavelength with the OD _{Photosensitive microsphere}/OD_{carrier body} ratio meeting the range of 0.85 to 1.15 is selected as lambda ₂, so that compared with the wavelength outside the range of the ratio, the value range of the concentration C ₂ of the photosensitive microsphere can be more accurately determined through the carrier concentration-absorbance curve of the carrier. Experiments show that when the wavelength lambda ₂ is in the range of 440 nm-580 nm, the ratio of the absorbance value OD _{Photosensitive microsphere} of the photosensitive microsphere to the absorbance value OD _{carrier body} of the carrier is within 0.85-1.15, which indicates that the content of the photosensitive substance has small influence on the measurement of the microsphere concentration, Otherwise, the method is reverse. In one embodiment, the wavelength lambda ₂ may be 440nm to 580nm. For example, the wavelength λ ₂ may be 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm. It should be understood that when the photosensitive materials selected by the photosensitive microspheres are different, the maximum characteristic peak of the photosensitive materials will be changed correspondingly under the influence of the properties of the photosensitive materials, and the wavelength lambda ₁ and the wavelength lambda ₂ are correspondingly adjusted, and the OD _λ1 and the OD _λ2 are correspondingly adjusted.

After determining the wavelength lambda ₂, a plurality of carriers with known different concentrations x and the same particle size can be scanned by adopting the same wavelength lambda ₂ to obtain a corresponding absorbance value y, so that an equation is established according to a formula (3) to calculate and obtain the values of k and b, and the concentration C ₂ of the photosensitive microsphere with the same particle size as the carrier can be calculated according to a formula (2). Further, the loading amount of carriers of different particle diameters at the same concentration to the photosensitive substance may be different, thereby affecting the absorbance value, i.e., the carrier concentration-absorbance curve is also related to the particle diameter of the carrier. Therefore, in order to establish an accurate and reliable carrier concentration-absorbance curve, in one embodiment, carriers with particle diameters within 190 nm-290 nm are selected to establish a corresponding carrier concentration-absorbance curve so as to control the deviation value between the calculated result of the concentration C ₂ of the photosensitive microsphere and the actual concentration to be within 10%. For example, the predetermined particle size of the carrier may be 190nm, 200nm, 220nm, 240nm, 260nm, 280nm or 290nm. For example, a corresponding carrier concentration-absorbance curve can be established using 190nm of carrier, thereby establishing an equation according to equation (3) to calculate the values for k and b. Preferably, C ₂ is selected from 10ug/ml to 200ug/ml.

Specifically, given that the known light sensing amount Ps ranges from 1.34 to 16.28, and k, b, and λ ₂ are known from the above calculation, conversely, the concentration C ₂ of the photosensitive microspheres can be adjusted according to formulas (1) and (2). That is, in the actual photo-excitation chemical detection process, after the photosensitive microsphere with unknown concentration is arbitrarily configured, under the condition that the specific value of the unknown concentration is unknown, the photosensitive microsphere with unknown concentration is scanned by the wavelength lambda ₁ and the wavelength lambda ₂ respectively to obtain corresponding absorbance values, namely OD _λ1 and OD _λ2, and then the specific value of the unknown concentration is calculated by the formula (2), if the value range of the unknown concentration falls within 10ug/ml to 200ug/ml, the calculated concentration C ₂ is substituted into the formula (1) to calculate the photosensitive amount Ps, and if the value of the Ps is between 1.34 and 16.28, the concentration of the configured photosensitive microsphere can be applied to photo-excitation chemical luminescence detection.

In summary, under the condition that the concentration C ₂ of the photosensitive microsphere is known and the value range is 10ug/ml to 200ug/ml, the photosensitive value Ps corresponding to the photosensitive microsphere can be obtained directly according to the formula (1) without using the formulas (2) and (3); similarly, the value of the photosensitive amount Ps corresponding to the photosensitive microsphere can be determined according to the above manner on the premise that the specific value of the photosensitive microsphere concentration C ₂ is unknown. If the calculated value of Ps is between 1.34 and 16.28, the photosensitive microsphere can achieve the above effect, namely the photosensitive microsphere can be applied to photo-excitation chemiluminescence detection, and the accuracy and precision of the detection result meet the clinical application requirements.

Further, in order to reduce the effect of substances other than the carrier and the photosensitive substance on the absorbance value, in one embodiment, the surface of the photosensitive microsphere is not coated with the polysaccharide; or the polysaccharide content of the photosensitive microsphere is not higher than 25mg per gram of mass. Wherein polysaccharide refers to carbohydrates containing three or more unmodified or modified monosaccharide units, such as dextran, starch, glycogen, inulin, levan, mannan, agarose, galactan, carboxydextran, aminodextran, and the like. The interference on the measurement result of the absorbance value is reduced by not adding the polysaccharide or controlling the content of the polysaccharide, so that the detection result in clinical application is more accurate.

Experimental raw material and equipment

TABLE 1

Material name	Storage conditions and expiration dates
		Luminescent particle (FG)	2-8Deg.C, sealed and light-proof
CEA antibody 1	≤-15℃
		CEA antibody 2	≤-15℃
Biotin	≤-15℃

TABLE 2

In the CEA detection kit and the application method thereof, the raw materials and the reagents used in the CEA detection kit can be purchased from the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 preparation of a luminescent concentrated solution

1. Dialyzing the raw materials, placing 1mg of antibody 1 in a 3.5KD dialysis bag, and dialyzing in 0.05M CB buffer (pH 9.6) for 3 times;

2. Concentration measurement, the protein recovered by dialysis was aspirated and transferred to a clean centrifuge tube, and the concentration was measured by BCA method and found to be 2.62mg/ml.

3. Particle treatment, taking 1ml of FG particles with the concentration of 10mg/ml in a centrifuge tube, centrifuging for 30 minutes, discarding the supernatant, carrying out ultrasonic resuspension by using 0.05M CB buffer, repeating the operation once, and fixing the volume of 50mg/ml for standby.

4. Coupling reaction, according to particles: mixing the particles and antigen protein with the mass ratio of the protein being 10:0.5, and carrying out coupling reaction by using a rotary mixing mode, wherein the rotary reaction is carried out for 20 hours; preparing 8mg/mL NaBH4 solution by using 0.05M CB buffer solution, adding the solution into each reaction tube immediately, and performing rotary reaction for 2 hours;

5. Constant volume, cleaning and constant volume: the centrifugation and washing were repeated twice to remove the excess protein, and finally the volume was fixed to 10mg/ml in the luminescent reagent buffer.

6. Preparing a luminous reagent, namely diluting the prepared luminous concentrated solution to 50ug/ml by using 50ml of luminous reagent buffer solution, and balancing for 2 hours for later use.

EXAMPLE 2 preparation of Biotin reagent

1. Raw material dialysis, respectively taking 1mg of antibody 2, respectively placing in 3.5KD dialysis bags, and dialyzing in 0.1MNaHCO3 buffer solution for 3 times;

2. Concentration measurement, the protein recovered by dialysis was aspirated and transferred to a clean centrifuge tube, and the concentration was measured by BCA method and found to be 1.48mg/ml.

3. Labeling reaction, preparing 20mg/ml biotin NHS active ester according to protein: labeling reaction is carried out by adopting a rotary mixing mode according to the biotin mol ratio of 1:30, and the reaction is carried out for 16 hours.

4. Dialysis of the marked product, product dialysis, dialysis bag specification: 3500D, dialysis buffer: 0.02MHEPES buffer, dialysis temperature: 2-8 ℃, dialysis time and times: 3 times of dialysis for 3 hours each time; the protein recovered by dialysis was aspirated and transferred to a clean centrifuge tube, and the protein concentration was measured using BCA method at 1.68mg/ml for use.

5. The biotin reagent is prepared, 50ml of biotin reagent buffer solution is used, and the prepared biotin concentrated solution is diluted to 1ug/ml and balanced for 2 hours for standby.

EXAMPLE 3 preparation of photosensitizing agent

1. Preparation of microspheres

Preparation of the (one) Carrier

A) A100 ml three-necked flask was prepared, 40mmol of styrene, 5mmol of acrolein, and 10ml of water were added to each of the three-necked flask, and after stirring for 10 minutes, N ₂ min was introduced into the three-necked flask.

B) 0.11g of ammonium persulfate and 0.2g of sodium chloride were weighed and dissolved in 40ml of water, respectively, to prepare aqueous solutions. The aqueous solution was added to the reaction system of the three-necked flask of step a), and N ₂ min was continued.

C) The reaction system was heated to 70℃and reacted for 15 hours to obtain an emulsion.

D) The emulsion after the reaction is cooled to room temperature and filtered by a suitable filter cloth. And washing the emulsion obtained after filtration by using deionized water through centrifugal sedimentation until the conductivity of the centrifuged supernatant approaches to that of the deionized water, diluting the emulsion by using water, and preserving the emulsion.

E) The average particle diameter of the gaussian distribution of the particle size of the latex microspheres in the emulsion was 190nm as measured by a nanoparticle sizer.

(II) preparation of photosensitive microspheres

A) A25 ml round bottom flask was prepared, 0.11g of copper phthalocyanine (i.e., photosensitive substance) was added, 10ml of N, N-dimethylformamide was stirred uniformly by magnetic force, and the round bottom flask was heated to 75℃in a water bath to obtain a photosensitive substance solution.

B) A 100ml three-necked flask was prepared, 10ml of 95% ethanol, 10ml of water and 10ml of the above 1.e) of the prepared carrier were added, and the mixture was stirred uniformly by magnetic force, and the three-necked flask was heated to 70℃in a water bath.

C) Slowly dripping the photosensitive substance solution in the step a) into the three-neck flask in the step b), stopping stirring after reacting for 2 hours at 70 ℃, and naturally cooling to obtain emulsion. It will be appreciated that the mass ratio of the carrier of step b) and the photosensitive material solution of step a) may be adjusted according to the requirements of the subsequent experiments.

D) Centrifuging the emulsion obtained in the step c) for 1 hour according to a centrifugal force of 30000G, discarding supernatant after centrifugation, and re-suspending by using 50% ethanol. After three repeated centrifugal washes, the microspheres were resuspended to the desired concentration in the subsequent experiments with 50mmol/L CB buffer at ph=10.

2. Method for determining value application range of photosensitive microsphere concentration C ₂

The experiment adopts the preset particle size to establish the linear relation between the carrier concentration and the absorbance value, so that the concentration C ₂ of the photosensitive microsphere is obtained according to the absorbance value OD _λ2 in the formula (2). This experiment is used to explain the establishment procedure of the above-described formula (2) and formula (3). 1. Full wavelength scanning and particle size detection of microspheres

Particle size detection was performed in advance to ensure uniformity of particle size of the microspheres used in the experiment. The main materials and equipment involved in the experiment are shown in table 3.

TABLE 3 Table 3

Raw materials and instruments	Specification and model	Manufacturer' s
			Carrier body	Particle diameter of 190nm	Boyang
Photosensitive microsphere	Particle diameter of 190nm	Boyang
			Mixing instrument	-	-
Particle diameter instrument	M0DEL380	PSS.NICOMP
			Ultraviolet spectrophotometer	UV-1600PC	MADAPA
Deionized water	-	-

The experimental process is specifically as follows:

1.1 selection of microsphere particle size

In this experiment, in order to ensure the consistency of data, subsequent experiments can be performed by uniformly using photosensitive microspheres and carriers having the same particle size, for example, 190 nm.

1.2 Preparation of different concentrations of Carrier and photosensitive microspheres

The prepared photosensitive microspheres and blank microspheres are respectively diluted by deionized water to prepare carriers and photosensitive microspheres with different concentrations, wherein the prepared concentrations are respectively 10ug/ml,20ug/ml,30ug/ml,40ug/ml,50ug/ml,60ug/ml,70ug/ml,80ug/ml,90ug/ml,100ug/ml and the like. Namely, respectively configuring 10 concentrations of carriers and 10 concentrations of photosensitive microspheres; in addition, 5ug/ml of photosensitive substance solution was prepared.

2. Microsphere particle size detection

The particle size meter was turned on and the particle size of the support and photosensitive microspheres of 20ug/ml was taken as an example for detection.

Wherein, experimental data are shown in fig. 1 and 2. The average particle diameter of the carrier is 187.1nm, and the average particle diameter of the photosensitive microsphere is 190.9nm.

The detection result of the particle size meter shows that the particle sizes of the carrier and the photosensitive microspheres are about 190nm, the wave crest is narrower, the particle sizes of the microspheres are more uniform, and the microspheres can be used as microspheres required by subsequent experiments.

3. Selecting wavelengths

Opening an ultraviolet spectrophotometer, preheating for 30min, adjusting the ultraviolet spectrophotometer, setting the wavelength to 300-800 nm, setting the step length to 1nm, calibrating zero by using deionized water, and sequentially detecting the photosensitive microspheres, the carrier and the photosensitive substance solution with the concentrations configured in the step 1.2. It is understood that the wavelength setting needs to be greater than 300nm because the scanned wavelength is susceptible to interference from absorbance values of other substances, thereby affecting the accuracy and precision of the detection result.

4. Experimental data

4.1 Photosensitive materials

As shown in FIG. 3, FIG. 3 shows the wavelength-absorbance curve of the photosensitive material after scanning at 300nm to 800 nm. As can be seen from fig. 3, the photosensitive dye has distinct peaks at 360nm, 610nm, 650nm and 680nm, respectively, wherein 680nm is the main peak, i.e. the maximum characteristic peak of the photosensitive substance.

4.2 Vectors

FIG. 4 is a graph showing the wavelength-absorbance curves of 10 carriers at different concentrations after scanning at 300nm to 800 nm. As can be seen from FIG. 4, the carriers with different concentrations have no characteristic peak in the graph after being scanned by visible light of 300nm to 800 nm. Meanwhile, as can be seen from fig. 4, the carriers with different concentrations have different absorbance values after scanning, and the microsphere concentration of the carrier is positively correlated with the absorbance value.

4.3 Photosensitive microspheres

Fig. 5 shows the absorbance curves of 10 photosensitive microspheres with different concentrations at the wavelength of 300 nm-800 nm, and as can be seen from fig. 5, the photosensitive microspheres with different concentrations show obvious peaks at 360nm, 610nm, 650nm and 680nm after being scanned by visible light with 300 nm-800 nm, wherein 680nm is the main peak, i.e. the maximum characteristic peak of the photosensitive microspheres is the same as the maximum characteristic peak of the photosensitive substance. I.e. the photosensitive substance filled with photosensitive microspheres will directly affect the wavelength value corresponding to the maximum characteristic peak. As can be seen from fig. 5, the absorbance values of the photosensitive microspheres at different concentrations after scanning are different, and the microsphere concentration of the photosensitive microspheres is positively correlated with the absorbance value. Thus, different concentrations of photosensitive microspheres affect the magnitude of the amount of light sensed Ps.

4.4 Determination of wavelength lambda ₁ and wavelength lambda ₂

The wavelength-absorbance curves of the same concentration of photosensitive microspheres and carrier were compared. As shown in FIG. 6, taking photosensitive microspheres and carriers with the concentration of 10 mug/ml as an example, after the carrier and the photosensitive microspheres with the same concentration scan the wavelength of 300 nm-800 nm, 680nm characteristic peak of the photosensitive microspheres is the maximum characteristic peak of the photosensitive substance. Therefore, by reading the absorbance value of 680nm to most reflect the content of the photosensitive substance in the photosensitive microsphere, the wavelength corresponding to the maximum characteristic peak can be selected as lambda ₁, namely lambda ₁ is 680nm. It can be understood that the photosensitive material sampled in the experiment is copper phthalocyanine, and when the photosensitive dye is other raw materials, the maximum characteristic peaks may be different, and the corresponding wavelength lambda ₁ is determined according to the actual situation.

For the determination of the wavelength lambda ₂, the purpose is to determine the concentration C ₂ of the photosensitive microspheres from the corresponding absorbance value OD _λ2. Therefore, for determining the concentration of the photosensitive microspheres based on the absorbance value, it is necessary to avoid the maximum characteristic peak of the photosensitive substance, that is, the wavelength λ ₂ is different from λ ₁. Such a design is because, when a region where a peak appears in the photosensitive substance is selected, the absorbance value at the wavelength corresponding to the peak contains the absorbance value of the carrier itself plus the absorbance value of the photosensitive substance, thereby affecting the concentration test of the photosensitive microsphere. As can be seen from FIG. 6, lambda ₂ is selected to be optimal between 400nm and 600nm, and no characteristic peak exists in the wavelength interval. Although the characteristic peak of the photosensitive substance does not exist in the wavelength range of 300-330 nm, the wavelength is easily influenced by protein substances in other samples to be tested, and the clinical application is influenced. The absorbance value corresponding to the wavelength of 700 nm-800 nm is low, so that the detection sensitivity is low, the fluctuation of the test result is large, and the absorbance value corresponding to the wavelength of 400 nm-600 nm is consistent, therefore, the wavelength lambda ₂ is selected from the wavelength of 400 nm-600 nm.

Further, in order to accurately determine the value of the wavelength lambda ₂, the absorbance of one of the photosensitive microspheres and the carrier with the same concentration is used for analysis. Photosensitive microspheres and carriers at a concentration of 50ug/ml were used as examples and are shown in Table 4.

To reduce the effect of the photoactive material on the concentration of the microspheres measured, the range of wavelengths lambda ₂ selected requires a ratio of OD _{Photosensitive microsphere}/OD_{carrier body} within 0.85 to 1.15 at the same wavelength, i.e., (1±15%). As can be seen from Table 4, the ratio of the absorbance value OD _{Photosensitive microsphere} of the photosensitive microsphere of 50ug/ml to the absorbance value OD _{carrier body} of the carrier of 50ug/ml is within 0.85 to 1.15 when the wavelength lambda ₂ is between 440nm and 580nm, thereby indicating that the absorbance value of the photosensitive substance corresponding to the wavelength in the interval has less influence on the absorbance values of the photosensitive microsphere and the carrier, and that the content of the photosensitive substance affects the determination of the concentration of the photosensitive microsphere when the ratio of OD _{Photosensitive microsphere}/OD_{carrier body} is greater than 1.15. Preferably, the ratio of OD _{Photosensitive microsphere}/OD_{carrier body} is 1.05 at wavelengths of 500nm and 510nm, the constant ratio indicating a constant effect of the photosensitive material. Therefore, in this embodiment, the wavelength lambda ₂ is preferably 500nm. It will be appreciated that when photosensitive materials of different materials are selected to prepare photosensitive microspheres, the wavelength lambda ₂ can be determined with reference to the above method.

TABLE 4 Table 4

5. Establishment of Carrier concentration-absorbance curve

In this embodiment, the carrier concentration-absorbance curve is established by selecting carriers with the same particle size and testing the absorbance values of different concentrations of the carriers at the wavelength lambda ₂.

After the wavelength lambda ₂ was determined in the above step 4.4, the experiment was conducted by selecting a carrier having a wavelength lambda ₂ of 500nm and a particle diameter of about 190nm, and the carrier concentration-absorbance curve is shown in FIG. 7.

Specifically, in order to obtain carriers with different concentrations, the mass of the carrier is obtained by a traditional drying method, deionized water is added into the carrier with known mass to prepare 10mg/ml of carrier, and the carrier is further diluted by the deionized water to prepare 10 kinds of carriers with the concentration of 10ug/ml,20ug/ml,30ug/ml,40ug/ml,50ug/ml,60ug/ml,70ug/ml,80ug/ml,90ug/ml,100ug/ml and the like. And scanning the carrier with each concentration by using a wavelength of 500nm to obtain an absorbance value OD ₅₀₀ corresponding to each concentration, and establishing a linear relation y=kx+b between the carrier concentration and the absorbance value. At a known concentration x, when lambda ₂ is 500nm, the absorbance y can be measured directly from the spectrophotometer, so that k of 0.0021 and b of 0.0359 can be calculated. After the values of k and b are determined, the carrier concentration x= (OD _λ2-b)/k＝(OD_λ2 -0.0359)/0.0021 is according to equation (2).

Since the above experiment obtained the values of k and b by measuring the absorbance values of different concentrations using only carriers having a particle diameter of 190nm, the applicant used carriers having different particle diameters for verification in order to verify the accuracy of the calculation result of the above carrier concentration x. Carriers with different particle sizes are prepared respectively, and the particle sizes of the carriers comprise 7 particle sizes such as 190nm,200nm,220nm,240nm,260nm,280nm and 300 nm. And preparing the carrier with each particle size into theoretical concentration values of 40ug/ml,50ug/ml and 60ug/ml according to the mass of the carrier calculated by a drying method. The absorbance value OD _λ2 of the carrier at each concentration of each particle size was measured at a wavelength of 500nm, given the theoretical concentration value. To obtain accurate results, each concentration of each particle size was divided into 3 parts for detection of absorbance values. After obtaining the absorbance value OD _λ2 corresponding to each concentration of each particle diameter of each carrier, the corresponding concentration value was calculated as (OD _λ2 -0.0359)/0.0021, and the calculated result was compared with the theoretical concentration value, and the deviation of the calculated result was determined. The results are shown in Table 5:

TABLE 5

As can be seen from the data in Table 5, when the particle diameter of the carrier is less than or equal to 280nm, the recovery deviation of the concentration is within 10%, that is, the deviation of the concentration value x of the carrier calculated according to (OD _λ2 -0.0359)/0.0021 from the theoretical concentration value is within 10%, which shows that the method for determining the concentration of the photosensitive microsphere according to the absorbance value adopted by the application has better accuracy. Thus, the values of k and b determined using the above method can be applied to the calculation of the concentration C ₂ of the photosensitive microspheres in the formula (2).

As can be seen from FIG. 7, in this experiment, the C ₂ has a good linear relationship when the value is 10ug/ml to 100 ug/ml. It should be noted that, the value of C ₂ is limited to 10ug/ml to 100ug/ml, and the value range of C ₂ can be further determined by combining the following experiments.

3. Comparing the influence of photosensitive microspheres with different mass ratios on the quantity of photosensitive light

1. Preparation of photosensitive microspheres with different mass ratios

According to the preparation method of the photosensitive microsphere in the first step and the second step, the corresponding photosensitive microsphere is prepared by adopting different mass ratios of the carrier to the photosensitive substance, namely, 6 photosensitive microspheres with the mass ratios of the carrier to the photosensitive substance of 10:4, 10:2, 10:1, 10:0.2, 10:0.04 and 10:0 are prepared firstly, namely, the microspheres 1 to 6 in the table 4 and the table 5. Wherein 10:0 represents that the photosensitive microsphere contains no photosensitive substance and is only an empty carrier. And then respectively diluting the prepared photosensitive microspheres with different mass ratios by deionized water, namely respectively diluting the prepared photosensitive microspheres corresponding to the six mass ratios by 500 times, 1000 times and 2000 times, wherein the photosensitive microspheres with each mass ratio obtain 3 diluted photosensitive microspheres with different concentrations. Scanning the diluted photosensitive microspheres by an ultraviolet spectrophotometer to obtain absorbance values OD corresponding to the wavelength lambda ₁ nm and the wavelength lambda ₂ nm, calculating according to the formula (2) to obtain a corresponding concentration value C ₂, and calculating according to the formula (2) to obtain a corresponding photosensitive quantity Ps. Specific data are shown in table 6 below. It is noted that, as shown in fig. 5, at the wavelength λ ₁, the photosensitive microsphere has a strong absorption peak, i.e., a maximum characteristic peak, and the absorbance value corresponding to the absorption peak is the most capable of reflecting the concentration of the photosensitive substance. The absorbance values of the photosensitive microspheres include the absorbance values of the carrier and the photosensitive material, and thus the true absorbance value OD _{λ1 Photosensitive material} of the photosensitive material is OD _{λ1 Photosensitive microsphere}-OD_{λ1 carrier body}.

TABLE 6

Further, corresponding average value of the sensitization amount and CV value of the variation coefficient are calculated according to each mass ratio of the sensitization amount of the sensitization materials with different dilution factors in the table 6, wherein CV value is the ratio of standard deviation to average value, and specific calculation results can refer to the related data in the table 7.

As can be seen from table 6, when the dilution factor is 500X, the calculated value of the concentration value C ₂ of the photosensitive microsphere 6 according to the formula (2) is 199, which is very close to the corresponding theoretical concentration value 198. Therefore, the range of C ₂ supplemented with the above experiment two, namely the range of C ₂ can be 10ug/ml to 200ug/ml.

TABLE 7

As can be seen from table 6, the photosensitive materials with the same mass ratio are more consistent in calculated photosensitive amount after being diluted by different multiples; as can be seen from table 7, the CV values of the light-sensitive amounts of the light-sensitive substances of different mass ratios were all within 10%, which indicates that the calculation results of the light-sensitive amounts determined according to the formulas (1) and (2) were less fluctuated and the calculation was more accurate. And the photosensitive substances with the same mass ratio are described, and the photosensitive quantity is related to the corresponding dilution factor, namely the concentration of the photosensitive microspheres.

Further, a graph of different mass fractions of the photosensitive material shown in fig. 8 obtained with the calculated photosensitive material can be plotted according to table 7. As can be seen from fig. 8, the correlation between the amount of light sensed by the photosensitive microsphere per unit concentration and the mass ratio of the photosensitive substance is detected based on the absorbance value, that is, the larger the mass ratio of the photosensitive substance is, the higher the concentration of the photosensitive substance is, the larger the amount of light sensed is. Meanwhile, when the mass ratio of the carrier to the photosensitive substance is smaller than 10:1, the linear relation between the photosensitive quantity and the photosensitive concentration is better; when the mass ratio of the carrier to the photosensitive material is 10 (2-4), the increase of the photosensitive quantity is obviously reduced, which indicates that the ratio of the photosensitive material, namely the quantity of the photosensitive material filled in the carrier is gradually increased to be saturated. Such trend changes correspond to the change in the amount of light actually sensed by the photosensitive microspheres. In addition, when the mass ratio of the carrier to the photosensitive substance is 10:4, the photosensitive quantity of the obtained photosensitive microsphere reaches the peak value of 20.12, and even if the mass ratio of the photosensitive substance is continuously improved, the photosensitive quantity of the photosensitive microsphere is not further increased, so that the material cost is saved by controlling the mass ratio of the carrier to the photosensitive substance.

Example 4 comparison of the Properties of photosensitive microspheres with different light-sensitive amounts in clinical applications

1. Preparing photosensitive reagents with different light-sensitive amounts according to the photosensitive microspheres with different light-sensitive amounts

A) And (3) photosensitive microsphere suspension treatment: sucking a certain amount of the photosensitive microspheres prepared in the step one and the step 2 in the embodiment 3, centrifuging in a high-speed refrigerated centrifuge, removing the supernatant, adding a certain amount of MES buffer, oscillating the microspheres on an ultrasonic cell disruption instrument by ultrasonic waves to re-suspend the microspheres, and adding the MES buffer to adjust the concentration of the photosensitive microspheres to 100mg/ml.

B) Avidin solution preparation: a quantity of streptavidin was weighed and dissolved to 8mg/ml in MES buffer.

C) Mixing: mixing the processed 100mg/ml photosensitive microsphere suspension, 8mg/ml avidin and MES buffer solution in the volume ratio of 2:5:1, and rapidly and uniformly mixing to obtain a reaction solution.

D) The reaction: 25mg/ml NaBH ₃ CN solution is prepared by adopting MES buffer solution, and the volume ratio of NaBH ₃ CN solution to reaction solution is rapidly and evenly mixed. The reaction was carried out at 37℃for 48 hours with rotation.

E) Closing: 75mg/ml Gly glycine solution and 25mg/ml NaBH ₃ CN solution are prepared by adopting MES buffer solution, and the Gly glycine solution, naBH ₃ CN solution and reaction solution are prepared according to the following ratio of 2:1:10 volume ratio, adding the mixed solution into the solution after the reaction in the step d), uniformly mixing, and rotating at the constant temperature of 37 ℃ for 2 hours. Then adding 200mg/ml BSA solution (MES buffer solution) and the mixed solution with the volume ratio of the reaction solution being 5:8, quickly mixing evenly, and carrying out rotary reaction at the constant temperature of 37 ℃ for 16 hours.

F) Cleaning: and e, adding MES buffer solution into the solution reacted in the step e, centrifuging by a high-speed refrigerated centrifuge, removing supernatant, adding fresh MES buffer solution, re-suspending by an ultrasonic method, centrifuging again, repeatedly washing for 3 times, suspending by a small amount of MES buffer solution, and determining the solid content to be 10mg/ml.

G) Preparing a photosensitive reagent: the universal buffer solution of the photosensitive reagent is used for preparing the photosensitive microspheres coated with streptavidin and adopting the 6 different photosensitive amounts in mass proportion, thereby preparing the photosensitive reagent with 6 different photosensitive amounts. The sensitization amount of the 6 sensitization agents is shown in table 8.

TABLE 8

Name of the name	Light sensing amount
		Photosensitive agent 1	0.77
Photosensitive agent 2	1.34
		Photosensitive agent 3	4.07
Photosensitive agent 4	11.49
		Photosensitive agent 5	16.28
Photosensitive agent 6	20.12

2. Evaluation of the Properties of the photosensitizing Agents of 6 different photosensitizers

The above 6 kinds of photosensitive reagents having different amounts of light are applied to the detection of clinical samples, thereby evaluating the basic performance of the photosensitive reagents having different amounts of light in the clinical application to the detection of samples.

Experimental raw materials and equipment:

TABLE 9

Instrument for measuring and controlling the intensity of light	Specification and model	Manufacturer' s
			LiCA detector	HT	Boyang biotechnology (Shanghai) Co., ltd
Hepatitis B surface antigen detection kit	HBsAg	Boyang biotechnology (Shanghai) Co., ltd

2.1 Testing the sensitivity of 6 different light-sensitive reagents

Samples cal1 to cal6 of the kit using 6 kinds of HBsAg having known different target molecule concentrations, and reagents 1 to 6 using 6 kinds of different amounts of the above prepared reagents were used. Firstly, each sample is respectively added into a corresponding reaction container, then a luminescent reagent and a biotin reagent are respectively added into each reaction container in sequence, and each reaction container is combined by incubation at 37 ℃ to form a first compound luminescent microsphere-antibody-antigen-antibody-biotin. And adding corresponding photosensitive reagents into each reaction container respectively, and carrying out photo-excitation chemiluminescence detection by using a LiCA detector to obtain corresponding chemiluminescence signal values. The data of each photosensitizing agent corresponding to the detected chemiluminescent signal values are shown in the third through eighth columns of Table 10. Wherein the target molecule concentration of the sample cal1 is 0, i.e. the sample contains no hepatitis B surface antigen, the sample cal1 is a negative sample, and the corresponding measured value can be used as the measuring standard of the signal value of each photosensitive reagent.

Table 10

Wherein the theoretical values in the first column in table 10 are the corresponding known target molecule concentrations in the 6 samples. The third through eighth columns in table 10 are chemiluminescent signal values measured in LiCA detectors for samples of various target molecule concentrations for each kit comprising a photosensitizing reagent and a luminescent reagent. According to the data in the table, for the samples with the same target molecule concentration, the light sensitivity of the light sensitive reagent is between 1.34 and 16.28, and the larger the light sensitivity is, the larger the data of the measured signal value is; when the light sensing amount of the photosensitive agent reaches 20.12, a signal drop phenomenon occurs instead. Therefore, 1.34 to 16.28 are selected as the range of the value of the light sensing amount Ps.

Further, as shown in table 11, for the same photosensing agent, as the ratio of signal values corresponding to samples with different target molecule concentrations, when the photosensing amount of the photosensing agent is lower than 1.34, the numerical distinction degree between the signal values corresponding to the photosensing agent 1 is very low, i.e. the detection sensitivity is low, and samples with different target molecules cannot be distinguished according to the signal values. While the corresponding signal value of the photosensitizing agent 2 is lower than that of the photosensitizing agents 3 to 6, a certain distinction can be made for target molecules of different concentrations. The corresponding signal values of the photosensitizing agents 3 to 6 show definite signal values for both low-concentration target molecules and higher-concentration target molecules; meanwhile, the same photosensitive reagent has obvious difference of signal value values corresponding to the target molecule concentrations with different sizes, and shows good differentiation, so that the concentration interval of the target molecules can be judged according to the signal values.

TABLE 11

Kit for detecting a substance in a sample	Photosensitive agent 1	Photosensitive agent 2	Photosensitive agent 3	Photosensitive agent 4	Photosensitive agent 5	Photosensitive agent 6
							cal2/cal1	2.43	1.25	2.21	2.25	2.21	1.92
cal6/cal1	10.57	1086.25	2220.23	1759.43	1705.22	1816.37

2.2 Testing the accuracy of the detection results of 5 photosensitive Agents with different amounts of light

Preparation of experimental samples:

Selecting target molecules with known same mass and diluting into three mass control samples sp1, sp2 and sp3 with known different concentrations; 10 samples S1 to S10 with target molecule concentrations decreasing linearly are selected, and the target molecules of all samples are HBsAg; 4 negative samples N1, N2, N3, N4 were selected without target molecule.

The total of 17 different samples were tested for target molecule concentration with 5 different amounts of photosensitizing reagents 2 to 6, respectively. The corresponding third to seventh columns of concentration data are obtained by converting the chemiluminescent signals detected by the LiCA detector, and as shown in table 12, the first column is referred to as the theoretical value, which is the true concentration value in each sample.

Table 12

Theoretical value	Sample of	Photosensitive agent 2	Photosensitive agent 3	Photosensitive agent 4	Photosensitive agent 5	Photosensitive agent 6
							0.02	sp1	0.0250	0.0235	0.0182	0.0186	0.0211
0.21	sp2	0.2512	0.2028	0.2056	0.2015	0.2062
							49.02	sp3	49.8386	51.1355	48.3229	50.1592	50.8613
106.85	S1	110.2827	112.8762	112.8134	115.0697	107.6863
							54.32	S2	57.5621	57.0891	57.5909	59.3762	60.6261
23.41	S3	24.9368	25.3758	23.9947	24.9785	24.5732
							7.37	S4	8.3332	8.3079	7.9536	8.1843	8.5471
2.50	S5	2.7045	2.7441	2.9045	2.9713	2.9642
							0.23	S6	0.2302	0.2549	0.2264	0.2332	0.2340
0.19	S7	0.2390	0.2256	0.2184	0.2289	0.2355
							0.10	S8	0.1351	0.0968	0.0811	0.0827	0.1033
0.0315	S9	0.0573	0.0357	0.0340	0.0352	0.0408
							0.0256	S10	0.0372	0.0268	0.0260	0.0268	0.0295
/	N1	0.0207	0.0170	0.0050	0.0051	0.0123
							/	N2	-0.0520	0.0144	-0.0020	-0.0030	-0.0103
/	N3	-0.0162	0.0093	0.0000	0.0000	-0.0017
							/	N4	0.0287	-0.0036	-0.0007	-0.0006	0.0059

Because the light-sensitive amount of the light-sensitive reagent 1 is too low, the experiment does not need to continue to adopt the light-sensitive reagent 1 to participate in the performance test of accuracy. From the test data in table 12, it can be seen that the higher the exposure amount of the exposure agent, the closer the test data to the theoretical value, i.e., the higher the accuracy. The light sensing amount of the light sensing reagent 2 is the lowest, the concentration value measured for the sample of the low concentration target molecule fluctuates greatly from the theoretical value, and the concentration value measured for the sample of the high concentration target molecule is closer to the theoretical value, so the light sensing amount of the light sensing reagent 2 can be regarded as the lower limit of the light sensing amount. That is, when the light-sensitive amount of the light-sensitive microspheres is less than 1.34, the target molecules with various concentrations cannot be accurately detected, which is unfavorable for meeting the clinical detection requirements.

2.3 Testing the accuracy of the detection results of 5 photosensitive Agents with different amounts of light

And continuously selecting samples sp1, sp2 and sp3 which are diluted to three known different concentrations and have target molecules with the same known mass in the 2.2, dividing the samples with each concentration into 10 parts, respectively testing with 5 photosensitive reagents to obtain corresponding concentration values, and obtaining a concentration Mean value Mean, standard deviation STDEV and variation coefficient CV of the 10 parts of samples. Specific values are shown in table 13.

TABLE 13

From the data in table 13, it is clear that the CV value of the same sample sp1 at the lowest concentration of the photosensitizing agent 2 is more than 10%, which means that the fluctuation of the test result is large and the accuracy is general, and therefore the photosensitizing amount 1.34 of the photosensitizing agent 2 can be used as the lower limit of the photosensitizing amount required for the photosensitizing microsphere in clinical detection.

Example 5 preparation of the kit

TABLE 14

Component name	Main composition of
		CEA reagent 1	CEA antibody coated luminescent microparticles
CEA reagent 2	Biotin-labeled CEA antibodies
		Photosensitive agent	Streptavidin coated with photosensitive microsphere prepared in example 3

Kits were prepared according to the compositions shown in table 14.

Example 6 methods of Using the kit

Scaling: the matched CEA calibrator is used for calibration, and the calibration is carried out by detecting a Rogowski assignment sample and controlling quality;

instrument: lica 500,500

The detection process is as shown in fig. 9:

The first step: taking 25ul of samples into the reaction holes;

And a second step of: sequentially adding a luminescent reagent and a biotin reagent into the reaction hole, so as to mix the luminescent reagent and the biotin reagent with the sample;

And a third step of: incubation, the immune reaction needs to be most fully combined with antigen-antibody at 37 ℃, and the luminous microsphere-antibody-antigen-antibody-biotin complex is more easily formed;

Fourth step: adding general liquid (photosensitive reagent 2-5 prepared in example 4), wherein the photosensitive amount is 1.34< PS <16.28, so that photosensitive sphere-SA in the general liquid is combined with biotin, if the photosensitive amount Ps of the photosensitive microsphere is less than 1.34, the photosensitive microsphere can not release enough singlet oxygen when excited by 680nm excitation light, and the singlet oxygen can be absorbed by other proteins in the liquid phase in the transfer process, so that the insufficient singlet oxygen can not be transferred to the luminescent microsphere, and can not be absorbed by the luminescent microsphere, and can not emit 610nm emission light; when the sensitization amount of the sensitization microsphere is 1.34< PS <16.28, the singlet oxygen energy can be ensured to be transferred to the luminous sphere and is absorbed by the luminous sphere to emit light of 610 nm;

Fifth step: the incubation is that the biotin-avidin combination is most sufficient for antigen-antibody combination at 37 ℃, so that the luminous microsphere-antibody-antigen-antibody-biotin-avidin-photosensitive microsphere complex is more easily formed; thereby the distance between the light sensing ball and the luminous ball is shortened;

Sixth step: reading, namely irradiating the reaction hole by using 680nm light, so that the photosensitive microsphere in the reaction hole emits singlet oxygen, when the distance between the photosensitive microsphere and the luminous sphere is smaller than 200nm, the luminous sphere can absorb the singlet oxygen, thereby emitting 610nm light, amplifying a signal by a PMT, and collecting a 610nm optical signal value; and (5) calculating the concentration through a calibration curve to obtain a concentration value.

Example 7

By using the CEA detection kit prepared in example 4, roche assignment samples were detected using 4 universal solutions (photosensitive reagents 2 to 5 prepared in example 4, respectively).

1. The calibration result is:

TABLE 15

2. Differentiation:

Table 16

The distinguishing degree shows that the general liquid 1 is low, and the distinguishing degree of the general liquid 2-4 is basically consistent; 3. rogowski assignment sample:

TABLE 17

The magnitude of the light sensing amount has less influence on the measured value of the clinical sample.

4. Quality control product precision:

TABLE 18

The precision results showed that the precision of general purpose liquid 1 was not as good as general purpose liquids 2 to 4. 2-4 weight-measuring CV of the universal liquid is within 10 percent, and the precision is good;

Conclusion: the photosensitive reagent with the photosensitive quantity of 1.34-16.28 can be suitable for the detection of the photo-activated chemiluminescence CEA kit, and the photo-activated chemiluminescence CEA kit with the photosensitive quantity of 4.07-16.28 has better performance.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The application of photosensitive microsphere in preparing microsphere composition, reagent combination, kit, detection system and/or detection device for detecting CEA is characterized in that,

The photosensitive microsphere comprises a carrier and a photosensitive substance carried by the carrier, wherein the photosensitive substance can generate singlet oxygen under the excitation of light; the photosensitive microsphere is prepared according to the mass ratio of the carrier to the photosensitive substance of 10 (0.04-4);

The sensitization amount Ps of the sensitization microsphere is between 1.34 and 16.28; the sensitization amount ps=od _λ1/C₂*10³, wherein:

The OD _λ1 is an absorbance value corresponding to a maximum absorption peak of a wavelength-absorbance curve obtained after full-wavelength scanning of the photosensitive microsphere with the concentration of C ₂ in a visible light region of 300-800 nm, and the lambda ₁ is a wavelength corresponding to a maximum absorption peak in the wavelength-absorbance curve obtained after full-wavelength scanning of the photosensitive substance in the visible light region of 300-800 nm; the unit of C ₂ is ug/ml, and the unit of C ₂ is the concentration of the photosensitive microsphere during the photo-excitation chemiluminescence detection;

concentration of the photosensitive microsphere

2. The use according to claim 1, wherein C ₂ is selected from 10ug/ml to 200ug/ml.

3. The use according to claim 1, wherein the linear relationship of carrier concentration-absorbance curve is y = kx+b, wherein:

4. The use according to claim 1, wherein the wavelength λ ₂ is selected from any wavelength value with a ratio of OD _{Photosensitive microsphere}/OD_{carrier body} within 0.85 to 1.15, and the wavelength λ ₂ is not equal to the wavelength λ ₁;

5. The method according to claim 4, wherein the wavelength lambda ₂ is 400nm to 600nm.

6. The use according to claim 1, wherein the photosensitive microspheres are carriers filled with photosensitive substances.

7. The use according to claim 1, wherein the wavelength λ ₁ is 600nm to 700nm.

8. The use according to any one of claims 1 to 7, wherein the carrier has a particle size of 190nm to 280nm.

9. The use according to any one of claims 1 to 7, wherein the surface of the photosensitive microsphere is not coated with a polysaccharide and the surface of the photosensitive microsphere is attached with an avidin selected from the group consisting of ovalbumin, vitellin, streptavidin, neutravidin and avidin-like molecules.

10. The use according to claim 9, wherein the avidin is streptavidin.

11. Microsphere composition for detection of CEA comprising luminescent microspheres and said photosensitive microspheres in the use according to any one of claims 1 to 10.

12. A reagent combination for detecting CEA, comprising a photosensitive reagent, a luminescent reagent, and a labeling reagent;

The photosensitizing agent comprising a buffer solution and the photosensitizing microspheres in the use of any one of claims 1 to 10 stored in the buffer solution;

the luminescent reagent comprises luminescent microspheres and substances capable of reacting with CEA;

the labeling reagent comprises a label and the substance capable of reacting with CEA;

the photosensitizing agent also includes a substance capable of binding to the label.

13. The combination of reagents according to claim 12, wherein the substance capable of reacting with CEA comprises: CEA antibodies;

And/or, the marker comprises: biotin;

And/or the substance capable of binding to the label comprises: streptavidin.

14. The reagent combination of claim 13, wherein the sugar content per liter of volume of the buffer solution is 1g ± 0.2g.

15. A kit for detecting CEA is characterized in that,

Comprises one or more of any of the following substances, acceptable auxiliary materials, auxiliary agents or carriers capable of reacting with CEA;

(I) The photosensitive microsphere in the use according to any one of claims 1 to 10; or (b)

(II) the microsphere composition of claim 11; or (b)

(III) the combination of reagents of any one of claims 12 to 14;

the carrier comprises: reagent bottles, reagent cards, test strips or chips;

The substance capable of reacting with CEA includes: CEA antibodies.

16. A system or device for detecting CEA, characterized in that,

Including one or more of any of the following;

(II) the microsphere composition of claim 11; or (b)

(III) the combination of reagents of any one of claims 12 to 14; or (b)

(IV) the kit of claim 15;

The substance capable of reacting with CEA includes: CEA antibodies.