Electrochemical immunosensor, and preparation method therefor and use thereof

Abstract

The present application belongs to the field of electrochemical immunosensors and the field of protein detection, and specifically relates to an electrochemical immunosensor, and a preparation method therefor and use thereof. Provided in the present application is an electrochemical immunosensor. The surface of an electrode is modified by using a carbon nanotube, which is modified in situ with gold nanoparticles, such that not only can the electron transport efficiency of the electrode be effectively improved, the effective working area of the surface of a sensor can also be increased, so as to provide a larger area for subsequent antibody immobilization. Furthermore, a modification film is self-assembled on the surfaces of the gold nanoparticles by means of Au-S bonds, such that the surface of the electrode is enriched with carboxyl groups, so as to effectively increase the number of carboxyl-group modifications per unit of area, and thus the ordered arrangement of antibodies can be achieved, thereby maximizing the immobilization efficiency of the antibodies. When the electrochemical immunosensor in the present application is used for detection, a detection signal can be effectively enhanced, the signal-to-noise ratio can be increased, and the detection limit can be lowered.

Description

Electrochemical immunosensor and preparation method and application thereof

[0001] Related applications

[0002] The present application claims priority to the Chinese patent application No. 202510061886.9, filed on January 15, 2025, entitled "Electrochemical immunosensor and preparation method and application thereof", the contents of which are hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of electrochemical immunosensor and protein detection, and particularly relates to an electrochemical immunosensor and a preparation method and application thereof. BACKGROUND

[0004] At present, in the medical field, common tumor markers include carcinoembryonic antigen (CEA), alpha-fetal protein (AFP), carbohydrate antigen 125 (CA125) and carbohydrate antigen 199 (CA199), etc. Carcinoembryonic antigen (CEA) is a non-specific tumor marker, and its normal level in human blood is 0 μg / L-5 μg / L. When it is higher than 20 μg / L, it may reflect the possibility of cancer in the body, such as colon cancer, pancreatic cancer, breast cancer and gastric cancer, etc. Therefore, the detection of cancer markers can be used as an important means for early cancer screening, and can also be used for monitoring the recurrence and metastasis of tumors.

[0005] At present, the main means for screening CEA is immunological method, which recognizes trace amounts of antigens in complex body fluids through high-specificity antigen-antibody affinity reaction, and then detects high enough signal through different signal amplification means. These methods include enzyme-linked immunosorbent assay, chemiluminescence, etc. However, these methods depend on precise large instruments, have complex detection processes and high cost, which limits their application.

[0006] In addition, in the early stage of cancer development, the change range of CEA level in the body is not large, so a high-specificity and high-sensitivity means is needed to detect such subtle changes. However, in the traditional technology, based on electrochemical sensor, there are still problems of low signal-to-noise ratio and high detection line in early detection of CEA. SUMMARY

[0007] Therefore, an embodiment of the present application provides an electrochemical immunosensor, which can effectively improve the signal-to-noise ratio of detection and reduce the detection line.

[0008] In one aspect, the present application provides an electrochemical immunosensor, which comprises an electrode substrate, and carbon nanotubes, gold nanoparticles and capture antibodies fixed on the surface of the electrode substrate in sequence.

[0009] The gold nanoparticles are fixed on the carbon nanotubes.

[0010] The gold nanoparticles are modified with a group with a carboxyl group at the end by forming an Au-S bond, and the carboxyl group is coupled with the capture antibody by forming a peptide bond.

[0011] In one of the embodiments, the capture antibody comprises one or more of CEA antibody, AFP antibody, CA125 antibody, and CA199 antibody.

[0012] In one of the embodiments, the electrode substrate comprises one or more of a screen-printed electrode, a glassy carbon electrode, and a magnetron sputtering electrode.

[0013] Another aspect of the present application provides a method for preparing an electrochemical immunosensor, comprising:

[0014] The carbon nanotubes are fixed on an electrode substrate to prepare a first carbon electrode.

[0015] Gold nanoparticles are modified on the first carbon electrode to prepare a second carbon electrode.

[0016] The second carbon electrode is modified with a carboxyl group, and the gold nanoparticles are modified with a group with a carboxyl group at the end by forming an Au-S bond to prepare a third carbon electrode.

[0017] The carboxyl group on the third carbon electrode is activated, and a capture antibody is coupled with the activated carboxyl group by forming a peptide bond to prepare an electrochemical immunosensor.

[0018] In one of the embodiments, the preparation of the first carbon electrode comprises:

[0019] A carbon nanotube dispersion solution is prepared, the carbon nanotube dispersion solution is coated on the surface of the electrode substrate, and drying treatment is performed to prepare the first carbon electrode.

[0020] In one of the embodiments, the concentration of carbon nanotubes in the carbon nanotube dispersion solution is 0.01wt% to 0wt%.

[0021] In one of the embodiments, the drying treatment comprises drying treatment with nitrogen.

[0022] In one of the embodiments, the drying treatment is performed for 1h to 2h.

[0023] In one of the embodiments, the preparation of the second carbon electrode comprises mixing a chloroauric acid solution with the first electrode, and using a chemical deposition method to prepare the second carbon electrode.

[0024] In one of the embodiments, the concentration of the chloroauric acid solution is 0.01wt% to 0.5wt%.

[0025] In one of the embodiments, the conditions of the chemical deposition method include a voltage of -0.8V to -0.1V and a time of 20s to 300s.

[0026] In one of the embodiments, the preparation of the third carbon electrode includes mixing the second carbon electrode with a mixed solution containing a mercapto compound and a carboxyl compound, reacting, and drying.

[0027] In one of the embodiments, the mixed solution containing a mercapto compound and a carboxyl compound includes one or more of mercaptosuccinic acid, 6-(mercaptomethyl)-dodecanedioic acid, and 1,3,5-pentanetricarboxylic acid, 3-(2-mercaptoethyl).

[0028] In one of the embodiments, the concentration of the mercaptosuccinic acid in the mixed solution is 5mM to 200mM.

[0029] In one of the embodiments, the concentration of the 6-(mercaptomethyl)-dodecanedioic acid in the mixed solution is 5mM to 200mM, respectively.

[0030] In one of the embodiments, the concentration of the 1,3,5-pentanetricarboxylic acid in the mixed solution is 5mM to 200mM.

[0031] In one of the embodiments, the concentration of the 3-(2-mercaptoethyl) is 5mM to 200mM.

[0032] In one of the embodiments, the reaction conditions include a temperature of 25℃ to 30℃ and a time of 12h to 24h.

[0033] In one of the embodiments, the drying process includes drying with nitrogen; and optionally, the time of the drying process is 1h to 2h.

[0034] In one of the embodiments, the activation process includes activating the third carbon electrode by dropping an activation solution.

[0035] The activation solution includes one or more of 2-(N-morpholino)ethanesulfonic acid, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0036] In one of the embodiments, the concentration of the 2-(N-morpholino)ethanesulfonic acid in the activation solution is 2mM to 40mM.

[0037] In one of the embodiments, the concentration of the N-hydroxysuccinimide is 5mg / mL to 30mg / mL.

[0038] In one of the embodiments, the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 5 mg / mL-30 mg / mL.

[0039] In one of the embodiments, the time of the activation treatment is 0.2 h-1 h.

[0040] Another aspect of the present application provides an antigen detection method, which uses the electrochemical immunosensor described above for detection.

[0041] In one of the embodiments, the method comprises: providing a detection antibody; the detection antibody is a HRP enzyme-labeled gold nanoparticle and antibody combination; adding the antigen to be detected to the surface of the electrochemical immunosensor for first incubation treatment.

[0042] After adding the detection antibody, second incubation is performed, color developing solution is added for reaction, electrochemical impedance is detected, and electrochemical impedance value is obtained.

[0043] In one of the embodiments, the conditions of the first incubation treatment comprise: temperature is 36.8℃-37.2℃, and time is 10 min-20 min.

[0044] In one of the embodiments, the conditions of the second incubation treatment comprise: temperature is 36.8℃-37.2℃, and time is 10 min-20 min.

[0045] In one of the embodiments, the color developing solution is a substrate of HRP enzyme.

[0046] In one of the embodiments, the time of the color developing solution reaction is 30 s-300 s.

[0047] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects and advantages of the present application will be apparent from the description and the claims thereof. BRIEF DESCRIPTION OF DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0049] Fig. 1 is a specific preparation process and reaction process of the electrochemical immunosensor of the present application;

[0050] Fig. 2 is the impedance test result of the electrode in different modification stages of the present application;

[0051] Figure 3 is a time-current curve test result of the electrochemical immunosensor of the present application;

[0052] Figure 4 is a linear range and detection limit result of the electrochemical immunosensor of the present application in measuring CEA antigen. DETAILED DESCRIPTION

[0053] The present application will be further described below in conjunction with embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not intended to limit the scope of the present application, and the purpose of providing these embodiments and examples is to make the disclosure of the present application more thoroughly and comprehensively understood. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms obtained thereby also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0055] Terminology

[0056] Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings:

[0057] The selection range of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0058] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0059] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0060] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0061] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0062] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0063] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0064] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0065] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0066] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0067] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0068] The term "CEA (carcinoembryonic antigen)" refers to carcinoembryonic antigen, which was first isolated from embryos and tumor tissues and is a broad-spectrum tumor marker.

[0069] The term "AuNP (Au nanoparticles)" refers to gold nanoparticles, a type of noble metal nanomaterial with a particle size of approximately 10-100 nm. These nanomaterials possess excellent electrical conductivity and high specific surface area, among other nanomaterial properties.

[0070] The term "CNTs (carbon nanotubes)" refers to carbon nanotubes, a type of carbon-based nanomaterial with varying diameters (10 nm to 200 nm) and lengths (1 μm to 10 μm), exhibiting excellent electrical conductivity and high specific surface area.

[0071] Electrochemical biosensors refer to sensors that immobilize biological recognition components (such as antigens, antibodies, enzymes, and nucleic acids) on their surface. These components recognize target substances, triggering a series of electrochemical reactions that alter the electrical signal. The sensitivity and efficiency of this system can be further improved by adding electroactive substances, redox probes, and nanomaterials.

[0072] Nanomaterials and enzyme-catalyzed substrate amplification strategies in electrochemical sensing. Enzyme-catalyzed substrate methods amplify antigen-antibody recognition into more detectable changes in electrical signals generated by electroactive products. Nanomaterials can increase antibody immobilization by increasing the sensor's specific surface area, and can also enrich nanomaterials with more enzymes for further amplification of the detection signal.

[0073] Enzyme-linked immunosorbent assay (ELISA) is a method that utilizes antigen-antibody interactions to detect target molecules. Antigens or antibodies are coated onto polystyrene plates, binding the target molecule. The antibody is then directly or indirectly coupled with an enzyme, proportionally converting the amount of the target molecule into the amount of enzyme and the amount of substrate catalyzed by the enzyme. A color reaction is then generated, and the target component is quantitatively analyzed by colorimetry.

[0074] Chemiluminescent immunoassay is a method for the quantitative detection of target proteins based on antigen-antibody binding. When the target protein forms an antigen-antibody complex with an antibody labeled with a tracer enzyme, the tracer enzyme catalyzes a chemical reaction in the luminescent substrate to produce light. The concentration of the target component can then be determined by detecting the amount of light emitted using an optical instrument.

[0075] This application provides an electrochemical immunosensor, including an electrode substrate, and carbon nanotubes, gold nanoparticles and capture antibodies sequentially immobilized on the surface of the electrode substrate.

[0076] In this method, gold nanoparticles are immobilized on carbon nanotubes using a chemical deposition method.

[0077] Gold nanoparticles are modified with carboxyl groups at the top end via Au-S bonds, and the carboxyl groups are coupled to the capture antibody via peptide bonds.

[0078] This application, by fixing carbon nanotubes on the electrode surface, can not only increase the effective working area of ​​the electrode, but also further improve the conductivity of the electrode, effectively enhancing the electron conduction efficiency between the electroactive material and the electrode through electrochemical reaction.

[0079] Next, gold nanoparticles were grown on the surface of carbon nanotubes via electrochemical deposition. This in-situ growth method can increase the bonding between gold nanoparticles and carbon nanotubes. Gold has a very high electron transport efficiency, and the modification with gold nanoparticles can further improve electrode performance. In addition, gold nanoparticles have a large specific surface area, which further increases the antibody binding sites on the electrode and effectively improves the antibody immobilization amount. Finally, the modification with gold nanoparticles allows for further modification of the electrode surface through Au-S bonds.

[0080] In a specific example, the capture antibody includes one or more of CEA antibody, AFP antibody, CA125 antibody, and CA199 antibody;

[0081] Optionally, the electrode substrate includes, but is not limited to, one or more of screen-printed electrodes, glassy carbon electrodes, and magnetron sputtering electrodes.

[0082] This application uses screen-printed electrodes as a substrate, upon which further electrode modifications are made to fabricate an electrochemical immunosensor. Screen-printed electrodes are low-cost, and the resulting sensors are disposable, reducing issues such as electrode contamination and surface passivation associated with repeated use. Precise customization and batch production offer excellent repeatability and stability, effectively reducing batch-to-batch variability.

[0083] Another aspect of this application provides a method for preparing an electrochemical immunosensor, comprising:

[0084] The first carbon electrode is prepared by fixing carbon nanotubes onto an electrode substrate;

[0085] A second carbon electrode was prepared by modifying the first carbon electrode with gold nanoparticles using a chemical deposition method.

[0086] The second carbon electrode was modified by carboxylation, which involved modifying gold nanoparticles with carboxyl groups at their ends by forming Au-S bonds, thus preparing the third carbon electrode.

[0087] The carboxyl groups on the third carbon electrode are activated, and then the capture antibody is coupled with the activated carboxyl groups by forming peptide bonds to prepare an electrochemical immunosensor.

[0088] In a specific example, the preparation of the first carbon electrode includes: preparing a carbon nanotube dispersion, coating the carbon nanotube dispersion onto the surface of the electrode substrate, and drying it to prepare the first carbon electrode.

[0089] Optionally, the concentration of the carbon nanotube dispersion is 0.01 wt% to 1.0 wt%; in one specific embodiment, the concentration of the carbon nanotube dispersion is 0.01 wt%, and in another specific embodiment, the concentration of the carbon nanotube dispersion is 1.0 wt%; it is understood that the concentration of the carbon nanotube dispersion is 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, and 0.9 wt% or other values.

[0090] Optionally, the drying process includes drying with nitrogen gas for 1 to 2 hours.

[0091] In one embodiment, the preparation of the second carbon electrode includes: mixing a chloroauric acid solution with the first electrode and preparing the second carbon electrode by chemical deposition.

[0092] Optionally, the concentration of the chloroauric acid solution is 0.01 wt% to 0.5 wt%; in one specific embodiment, the concentration of the chloroauric acid solution is 0.01 wt%, and in another specific embodiment, the concentration of the chloroauric acid solution is 0.5 wt%; it is understood that the concentration of the chloroauric acid solution is 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, and 0.5 wt% or other values.

[0093] Optionally, the conditions for chemical deposition include a voltage of -0.8V to 0.2V and a time of 20s to 300s. In one specific embodiment, the voltage is -0.8V, and in another specific embodiment, the voltage is -0.1V; it is understood that the voltage can also be -0.7V, -0.6V, -0.5V, -0.4V, -0.3V, and -0.2V, as well as other values.

[0094] In one embodiment, the preparation of the third carbon electrode includes: mixing the second carbon electrode with a mixture containing a thiol compound and a carboxyl compound, reacting the mixture, and then drying it.

[0095] Optionally, the mixture containing thiol compounds and carboxyl compounds includes: mercaptosuccinic acid, 6-(mercaptomethyl)-dodecanoic acid and 1,3,5-pentanetricarboxylic acid, 3-(2-mercaptoethyl).

[0096] In one specific example, the concentration of mercaptosuccinic acid in the mixture is 5 mM to 200 mM.

[0097] For example, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, or 200mM.

[0098] Optionally, the concentration of 6-(mercaptomethyl)-dodecanoic acid in the mixture is 5 mM to 200 mM. For example, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM.

[0099] Optionally, the concentration of 1,3,5-pentanetricarboxylic acid in the mixture is 5 mM to 200 mM. For example, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM.

[0100] The carboxyl groups modified on the electrode surface are activated by N-hydroxysuccinimide solution and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, enabling them to undergo a condensation reaction with the amino group of the antibody, thus coupling the antibody to the electrode. Finally, excess active sites are blocked to prevent non-specific adsorption.

[0101] Further optionally, the concentration of 3-(2-mercaptoethyl) is 5 mM to 200 mM. For example, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM.

[0102] In a specific example, the reaction conditions include: a temperature of 25℃ to 30℃ and a time of 12h to 24h; for example, temperatures of 25℃, 26℃, 27℃, 28℃, 29℃, and 30℃, and times of 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, and 24h, as well as other values.

[0103] In a specific example, the drying process includes drying with nitrogen gas for 1 to 2 hours.

[0104] Optionally, the activation treatment includes: activating the third carbon electrode by adding an activation solution;

[0105] The activating solution includes 2-(N-morpholino)ethanesulfonic acid, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;

[0106] Optionally, the concentration of 2-(N-morpholino)ethanesulfonic acid is 2 mM to 40 mM; in one specific embodiment, the concentration of 2-(N-morpholino)ethanesulfonic acid is 2 mM, and in another specific embodiment, the concentration of 2-(N-morpholino)ethanesulfonic acid is 40 mM; it is understood that the concentration of 2-(N-morpholino)ethanesulfonic acid is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, or other values.

[0107] Optionally, the concentration of N-hydroxysuccinimide is 5 mg / mL to 30 mg / mL; in one specific embodiment, the concentration of N-hydroxysuccinimide is 5 mg / mL, and in another specific embodiment, the concentration of N-hydroxysuccinimide is 30 mg / mL; it is understood that the concentration of N-hydroxysuccinimide is 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or other values.

[0108] Optionally, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 5 mg / mL to 30 mg / mL. In one specific embodiment, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 5 mg / mL, and in another specific embodiment, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 30 mg / mL; it is understood that the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or other values.

[0109] Optionally, the activation treatment time is 0.2h to 1h. For example, the time is 0.2h, 0.3h, 0.4h, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, or 1.0h.

[0110] In a specific example, the capture antibody is one or more of CEA antibody, AFP antibody, CA125 antibody, and CA199 antibody.

[0111] Optionally, the concentration of CEA antibody is 5 μg / mL to 125 μg / mL. In one specific embodiment, the concentration of CEA antibody is 5 μg / mL, and in another specific embodiment, the concentration of CEA antibody is 125 μg / mL; it is understood that the concentration of CEA antibody can be 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, or other values.

[0112] This application also provides an antigen detection method that uses the aforementioned electrochemical immunosensor for detection.

[0113] In a specific example, this includes: providing a detection antibody; the detection antibody being a conjugate of HRP-labeled gold nanoparticles and the antibody.

[0114] The antigen to be tested is dropped onto the surface of the electrochemical immunosensor for the first incubation treatment.

[0115] After adding the detection antibody, a second incubation is performed, followed by the addition of a colorimetric solution to initiate the reaction. The electrochemical impedance is then measured to obtain the electrochemical impedance value.

[0116] After the immunosensor binds to the antigen, HRP-labeled detection antibodies are added for characterization. When the detection antibody binds to the electrode via an antigen-antibody reaction, it increases the HRP content on the electrode surface. After the substrate is added, HRP catalyzes the substrate to generate an electroactive product, which undergoes a redox reaction at a specific voltage, resulting in a change in current.

[0117] In antibody labeling detection, gold nanoparticles are used as carriers to load as much HRP enzyme as possible before labeling the antibody. Compared to directly labeling HRP on the antibody, loading a large amount of HRP onto nanoparticles effectively increases the HRP loading per unit concentration of antibody, thereby increasing the amount of product and ultimately leading to a larger current change, thus effectively improving the sensor's sensitivity.

[0118] In a specific example, the conditions for the first incubation treatment include a temperature of 36℃ to 38℃ and a time of 10 min to 20 min. For example, the temperature is 36℃, 37℃, or 38℃, and the time is 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min.

[0119] In a specific example, the conditions for the second incubation treatment include a temperature of 36℃ to 38℃ and a time of 10 min to 20 min. For example, the temperature is 36℃, 37℃, or 38℃, and the time is 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min.

[0120] In one specific example, the chromogenic solution comprises a substrate for HRP enzyme.

[0121] In a specific example, the reaction time for adding the colorimetric solution is 30s to 300s. For example, the time is 30s, 50s, 70s, 90s, 110s, 130s, 150s, 170s, 190s, 210s, 230s, 250s, 270s, 290s, and 300s.

[0122] This application provides an electrochemical immunosensor. By modifying the electrode surface with carbon nanotubes in situ using gold nanoparticles, the electron transport efficiency of the electrode is effectively improved, and the effective working area of ​​the sensor surface is increased, providing a larger area for subsequent antibody immobilization. Furthermore, a modified film is formed on the gold nanoparticle surface through Au-S bonds, enriching the electrode surface with carboxyl groups. This effectively increases the number of carboxyl groups per unit area, enabling the orderly arrangement of antibodies and maximizing antibody immobilization efficiency. When using the electrochemical immunosensor of this application for detection, the detection signal is effectively improved, the signal-to-noise ratio is increased, and the detection limit is reduced.

[0123] Furthermore, when this application is used for testing, the sample does not require processing, the detection process is shorter and the time is shorter, which can make the detection more efficient and improve the sensitivity. In the future, it can be automated by designing microfluidic chips, which can further reduce manual operation, achieve high-throughput testing, and facilitate the development of POCT.

[0124] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0125] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0126] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0127] Example 1

[0128] This embodiment provides a method for preparing an electrochemical immunosensor. The specific preparation process and reaction process are shown in Figure 1, and include:

[0129] 1. Preparation of CNT dispersion

[0130] Chitosan was weighed and added to a 0.05M sodium acetate buffer solution with pH 5.0. After stirring thoroughly, a chitosan solution with a concentration of 2.5wt% was prepared. Then, carbon nanotube powder was added to the solution and ultrasonically dispersed at 300W for 3 hours to obtain a black CNT dispersion with a concentration of 0.5wt%.

[0131] 2. Modification of CNTs (first electrode)

[0132] Screen-printed electrodes were selected for fabricating the sensor, using carbon electrodes as the working and counter electrodes, and an Ag / AgCl electrode as the reference electrode. A CNT dispersion was drop-coated onto the clean surface of the working electrode and dried under an infrared lamp. After gently rinsing with PBS solution, the electrode surface was dried with nitrogen gas to obtain a CNT-modified carbon electrode (C-CNT electrode).

[0133] 3. AuNP modification (second electrode)

[0134] A 0.25% chloroauric acid solution was added to the electrode region of the SPCE, and AuNPs were deposited on the surface of the working electrode C-CNTs electrode at a voltage of 0.2V using a chronoamperometry method for 250s, resulting in a carbon electrode with dual AuNP and CNTs modification (C-CNTs / AuNP electrode).

[0135] 4. Modification of carboxyl groups (third electrode)

[0136] The screen-printed electrode was treated with 150 mM mercaptosuccinic acid and reacted overnight at room temperature to allow it to spontaneously form a carboxyl-rich self-assembled film on the surface of gold nanoparticles via Au-S bonds. The electrode was then gently washed with PBS solution and dried with nitrogen gas.

[0137] 5. Activation of the carboxyl group

[0138] Equal volumes of a mixture of 25 mM 2-(N-morpholino)ethanesulfonic acid solution, 20 mg / mL N-hydroxysuccinimide solution, and 20 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution were mixed. 25 μL of this mixture was then added dropwise to a C-CNTs / AuNP electrode and activated at room temperature for 1 h. After the electrode was semi-dry, 25 μL of 85 μg / mL CEA antibody was added to the working electrode surface. The electrode was then placed in a humidified chamber at 37°C for 2 h to allow the amino groups on the antibody to condense with the activated carboxyl groups on the self-assembled membrane, thus coupling the antibody. The electrode was then washed with PBST to remove unstable CEA antibody, yielding the C-CNTs / AuNP-Ab electrode.

[0139] 6. Electrode sealing

[0140] The C-CNTs / AuNP-Ab electrode was immersed in a solution containing 5 wt% BSA and sealed at 37°C for 1.5 h. The electrode was then cleaned and dried before use.

[0141] 7. Preparation of gold nanoparticles

[0142] Gold nanoparticles were prepared using a reduction method. A 0.25 wt% chloroauric acid solution was heated to 100 °C, and an appropriate amount of trisodium citrate was added during stirring to bring the final concentration in the mixed solution to 2.5 wt%. The reaction continued to produce a red solution, which was then heated to 100 °C and boiled for 25 min. After cooling to room temperature, deionized water was added to prepare a 1% gold nanoparticle solution.

[0143] 8. Gold nanoparticle-labeled antibodies and HRP enzyme

[0144] The pH of 5 mL of gold nanoparticle solution was adjusted to 7 using K2CO3. Then, 0.25 mg of CEA monoclonal antibody and 1 mg of HRP enzyme were added, thoroughly mixed, and incubated at room temperature for 3 h. Afterward, 20% wt BSA solution was added to block the gold nanoparticle surface. The solution was centrifuged at 12000 rpm for 20 min and washed three times to obtain HRP-rich gold nanoparticle-labeled CEA antibody (AuNP / HRP-Ab).

[0145] Example 2

[0146] This embodiment provides a method for preparing an electrochemical immunosensor, including:

[0147] 1. Preparation of CNT dispersion

[0148] Chitosan was weighed and added to a 0.01M sodium acetate buffer solution with pH 4.0. After stirring thoroughly, a chitosan solution with a concentration of 0.1wt% was prepared. Then, carbon nanotube powder was added to the solution and ultrasonically dispersed at 300W for 2 hours to obtain a black CNT dispersion with a concentration of 0.01wt%.

[0149] 2. Modification of CNTs (first electrode)

[0150] Screen-printed electrodes were selected for fabricating the sensor, using carbon electrodes as the working and counter electrodes, and an Ag / AgCl electrode as the reference electrode. A CNT dispersion was drop-coated onto the clean surface of the working electrode and dried under an infrared lamp. After gently rinsing with PBS solution, the electrode surface was dried with nitrogen gas to obtain a CNT-modified carbon electrode (C-CNT electrode).

[0151] 3. AuNP modification (second electrode)

[0152] A 0.01% chloroauric acid solution was added to the electrode region of the SPCE, and AuNPs were deposited on the surface of the working electrode C-CNTs electrode at a voltage of -0.8V using a chronoamperometry method for 20s, resulting in a carbon electrode with dual AuNP and CNTs modification (C-CNTs / AuNP electrode).

[0153] 4. Modification of carboxyl groups (third electrode)

[0154] The screen-printed electrode was treated with a 150 mM aqueous solution of 6-(mercaptomethyl)-dodecanoic acid and reacted overnight at room temperature. This allowed the electrode to spontaneously form a carboxyl-rich self-assembled film on the surface of gold nanoparticles via Au-S bonds. The electrode was then gently washed with PBS solution and dried with nitrogen gas.

[0155] 5. Activation of the carboxyl group

[0156] Equal volumes of a 2 mM 2-(N-morpholino)ethanesulfonic acid solution, a 5 mg / mL N-hydroxysuccinimide solution, and a 20 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution were mixed. 5 μL of this mixture was then added dropwise to a C-CNTs / AuNP electrode and activated at room temperature for 0.2 h. After the electrode was semi-dry, 5 μL of a 5 μg / mL CEA antibody was added to the working electrode surface. The electrode was then placed in a humidified chamber at 37°C for 1 h to allow the amino groups on the antibody to condense with the activated carboxyl groups on the self-assembled membrane, thus coupling the antibody. The electrode was then washed with PBST to remove any unstable CEA antibody, yielding the C-CNTs / AuNP-Ab electrode.

[0157] 6. Electrode sealing

[0158] The C-CNTs / AuNP-Ab electrode was immersed in a solution containing 5 wt% CAS and sealed at 37°C for 1 hour. The electrode was then cleaned and dried before use.

[0159] 7. Preparation of gold nanoparticles

[0160] Gold nanoparticles were prepared using a reduction method. A 0.01 wt% chloroauric acid solution was heated to 100°C, and an appropriate amount of sodium citrate solution was added during stirring to bring the final concentration in the mixed solution to 1 wt%. The reaction continued to produce a red solution, which was then heated to 100°C and boiled for 5 minutes. After cooling to room temperature, deionized water was added to prepare a 1% gold nanoparticle solution.

[0161] 8. Gold nanoparticle-labeled antibodies and HRP enzyme

[0162] The pH of 5 mL of gold nanoparticle solution was adjusted to 6 using K2CO3. Then, 0.02 mg of CEA monoclonal antibody and 0.1 mg of HRP enzyme were added, thoroughly mixed, and incubated at room temperature for 2 h. Afterward, 20% wt BSA solution was added to block the gold nanoparticle surface. The solution was centrifuged at 8000 rpm for 5 min and washed three times to obtain HRP-rich gold nanoparticle-labeled CEA antibody (AuNP / HRP-Ab).

[0163] Example 3

[0164] This embodiment provides a method for preparing an electrochemical immunosensor, including:

[0165] 1. Preparation of CNT dispersion

[0166] Chitosan was weighed and added to a 0.1M sodium acetate buffer solution with pH 6.0. After stirring thoroughly, a chitosan solution with a concentration of 5.0 wt% was prepared. Then, carbon nanotube powder was added to the solution and ultrasonically dispersed at 300W for 4 hours to obtain a black CNT dispersion with a concentration of 1.0 wt%.

[0167] 2. Modification of CNTs (first electrode)

[0168] Screen-printed electrodes were selected for fabricating the sensor, using carbon electrodes as the working and counter electrodes, and an Ag / AgCl electrode as the reference electrode. A CNT dispersion was drop-coated onto the clean surface of the working electrode and dried under an infrared lamp. After gently rinsing with PBS solution, the electrode surface was dried with nitrogen gas to obtain a CNT-modified carbon electrode (C-CNT electrode).

[0169] 3. AuNP modification (second electrode)

[0170] A 0.5% chloroauric acid solution was added to the electrode region of the SPCE, and AuNPs were deposited on the surface of the working electrode C-CNTs electrode at a voltage of 0.2V using a chronoamperometry method for 300s, resulting in a carbon electrode with dual AuNP and CNTs modification (C-CNTs / AuNP electrode).

[0171] 4. Modification of carboxyl groups (third electrode)

[0172] The screen-printed electrode was treated with a 200 mM aqueous solution of 3-(2-mercaptoethyl) and reacted overnight at room temperature. This allowed the electrode to spontaneously form a carboxyl-rich self-assembled film on the surface of gold nanoparticles via Au-S bonds. The electrode was then gently washed with PBS solution and dried with nitrogen gas.

[0173] 5. Activation of the carboxyl group

[0174] Equal volumes of a mixture of 40 mM 2-(N-morpholino)ethanesulfonic acid solution, 30 mg / mL N-hydroxysuccinimide solution, and 30 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution were mixed. 50 μL of this mixture was then added dropwise to a C-CNTs / AuNP electrode and activated at room temperature for 1 h. After semi-drying, 50 μL of 125 μg / mL CEA antibody was added to the working electrode surface. The electrode was then placed in a humidified chamber at 37°C for 4 h to allow the amino groups on the antibody to condense with the activated carboxyl groups on the self-assembled membrane, thus coupling the antibody. The electrode was then washed with PBST to remove unstable CEA antibody, yielding the C-CNTs / AuNP-Ab electrode.

[0175] 6. Electrode sealing

[0176] The C-CNTs / AuNP-Ab electrode was immersed in a solution containing 10 wt% PEG and sealed at 37°C for 2 hours. The electrode was then cleaned and dried before use.

[0177] 7. Preparation of gold nanoparticles

[0178] Gold nanoparticles were prepared using a reduction method. A 0.5 wt% chloroauric acid solution was heated to 100°C, and an appropriate amount of tannic acid solution was added during stirring to bring the final concentration in the mixed solution to 5 wt%. The reaction continued to produce a red solution, which was then heated to 100°C and boiled for 30 min. After cooling to room temperature, deionized water was added to prepare a 1% gold nanoparticle solution.

[0179] 8. Gold nanoparticle-labeled antibodies and HRP enzyme

[0180] The pH of 5 mL of gold nanoparticle solution was adjusted to 6 using K2CO3. Then, 0.5 mg of CEA monoclonal antibody and 2 mg of HRP enzyme were added, thoroughly mixed, and incubated at room temperature for 6 h. Afterward, 20% wt BSA solution was added to block the gold nanoparticle surface. The solution was centrifuged at 15000 rpm for 30 min and washed three times to obtain HRP-rich gold nanoparticle-labeled CEA antibody (AuNP / HRP-Ab).

[0181] CEA antigen test results:

[0182] CEA antigen at gradient concentrations was prepared using antibody dilution buffer and added to the prepared C-CNTs / AuNP-Ab electrode surface. After incubation at 37°C for 15 min, the electrode was washed with PBST, and AuNP / HRP-Ab solution was added and incubated at 37°C for 15 min. After washing with PBST, TMB solution was added and the reaction was allowed to proceed for 300 s. The current was then measured at 0.1 V using chronoamperometry.

[0183] Impedance tests were performed on the electrodes at the different modification stages described above. A solution containing 5 mM potassium ferricyanide and 5 mM potassium chloride was used as the electrolyte solution. Impedance spectroscopy was performed at an open-circuit voltage with amplitude modulation of 0.01 V. The test results are shown in Figure 2.

[0184] The results showed that the electrode impedance decreased significantly after modification with CNTs and AuNPs, because both have good conductivity and increase the electrode surface area, which is beneficial to the electrochemical reaction between potassium ferricyanide and the electrode surface. After the electrode was modified and activated by the linker, the impedance increased significantly after antibody coupling. This was because the increase of non-conductive biological components on the electrode surface hindered electron transport between potassium ferricyanide and the electrode, indicating that this application can effectively immobilize a large number of antibody proteins.

[0185] Furthermore, the nanomaterial modification on the electrode increased the antibody binding region, and the polycarboxyl compounds modified on the nanomaterials resulted in a higher number of carboxyl groups per unit area on the electrode, further promoting antibody binding. After blocking, the impedance value increased further, and the surface electrode surface was covered with more non-conductive components, indicating that many non-specific sites on the electrode were blocked.

[0186] Detection limit test:

[0187] The electrochemical immunosensor designed in this application was used to detect gradient concentrations of CEA antigen (0, 0.1 ng / mL, 1.0 ng / mL, 10 ng / mL, 20 ng / mL). The time-current curves are shown in Figure 3. The current value at 60 s was statistically analyzed, and the current signal was plotted as a function of the antigen concentration.

[0188] As shown in Figure 3, the current value increases with the increase of CEA antigen concentration. This is because the number of antigens bound to the capture antibody increases, resulting in more detection antibodies binding to the electrode surface. The detection antibody is connected to HRP-rich gold nanoparticles, which, upon the addition of the substrate, allow more HRP to exert a catalytic effect, generating electroactive products and producing a higher current signal under the same voltage.

[0189] As shown in Figure 4, the electrochemical immunosensor exhibits a linear range of 0.1 ng / mL to 20 ng / mL when measuring CEA antigen, with an R² of 0.997 and a detection limit of 0.04 ng / mL.

[0190] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. An electrochemical immunosensor, characterized in that, It includes an electrode substrate, and carbon nanotubes, gold nanoparticles and capture antibodies sequentially immobilized on the surface of the electrode substrate; The gold nanoparticles are fixed on the carbon nanotubes; The gold nanoparticles are modified with Au-S bonds to form carboxyl groups at their ends, and the carboxyl groups are coupled to the capture antibody by forming peptide bonds.

2. The electrochemical immunosensor according to claim 1, characterized in that, The capture antibody includes one or more of CEA antibody, AFP antibody, CA125 antibody and CA199 antibody; Optionally, the electrode substrate includes one or more of screen-printed electrodes, glassy carbon electrodes, and magnetron sputtering electrodes.

3. A method for preparing an electrochemical immunosensor, characterized in that, include: The carbon nanotubes are fixed onto an electrode substrate to prepare a first carbon electrode; A second carbon electrode is prepared by modifying the first carbon electrode with gold nanoparticles. The second carbon electrode is carboxylated, wherein the carboxylation modification is performed by forming Au-S bonds to modify gold nanoparticles with carboxyl-terminated groups to prepare a third carbon electrode; and The carboxyl groups on the third carbon electrode are activated, and then the captured antibody is coupled to the activated carboxyl groups by forming peptide bonds to prepare an electrochemical immunosensor.

4. The method for preparing the electrochemical immunosensor according to claim 3, characterized in that, The preparation of the first carbon electrode includes: A carbon nanotube dispersion is prepared, and the carbon nanotube dispersion is coated onto the surface of the electrode substrate and dried to prepare a first carbon electrode. Optionally, the concentration of carbon nanoparticles in the carbon nanotube dispersion is 0.01 wt% to 1.0 wt%. Optionally, the drying process includes drying with nitrogen gas; Optionally, the drying time is 1 to 2 hours.

5. The method for preparing the electrochemical immunosensor according to claim 3, characterized in that, The preparation of the second carbon electrode includes: A second carbon electrode was prepared by mixing a chloroauric acid solution with the first electrode and using a chemical deposition method. Optionally, the concentration of the chloroauric acid solution is 0.01 wt% to 0.5 wt%. Optionally, the conditions for chemical deposition include: a voltage of -0.8V to -0.1V and a time of 20s to 300s.

6. The method for preparing the electrochemical immunosensor according to claim 3, characterized in that, The preparation of the third carbon electrode includes: The second carbon electrode is mixed with a mixture containing thiol and carboxyl compounds, reacted, and then dried. Optionally, the mixture containing thiol and carboxyl compounds includes one or more of the following: mercaptosuccinic acid, 6-(mercaptomethyl)-dodecanoic acid, 1,3,5-pentanetricarboxylic acid, and 3-(2-mercaptoethyl).

7. The method for preparing the electrochemical immunosensor according to claim 6, characterized in that, The method has one or more of the following conditions: (1) The concentration of the mercaptosuccinic acid in the mixture is 5 mM to 200 mM; (2) The concentrations of the 6-(mercaptomethyl)-dodecanoic acid in the mixture are 5 mM to 200 mM, respectively. (3) The concentration of the 1,3,5-pentanetricarboxylic acid in the mixture is 5 mM to 200 mM; (4) The concentration of 3-(2-mercaptoethyl) is 5 mM to 200 mM; (5) The reaction conditions include: a temperature of 25℃~30℃ and a time of 12h~24h; and, (6) Drying treatment includes: drying with nitrogen gas; Optionally, the drying time is 1 to 2 hours.

8. The method for preparing the electrochemical immunosensor according to claim 3, characterized in that, Activation treatment includes: The third carbon electrode was activated by adding an activation solution. The activation solution includes one or more of 2-(N-morpholino)ethanesulfonic acid, N-hydroxysuccinimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; Optionally, the concentration of 2-(N-morpholino)ethanesulfonic acid in the activation solution is 2 mM to 40 mM; Optionally, the concentration of the N-hydroxysuccinimide is 5 mg / mL to 30 mg / mL; Optionally, the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 5 mg / mL to 30 mg / mL; Optionally, the activation treatment time is 0.2h to 1h.

9. An antigen detection method, characterized in that, The detection was performed using the electrochemical immunosensor as described in any one of claims 1 to 2.

10. The antigen detection method according to claim 9, characterized in that, include: Provide detection antibodies; the detection antibodies are HRP-labeled gold nanoparticles conjugates with antibodies; The antigen to be tested is dropped onto the surface of the electrochemical immunosensor for the first incubation treatment. as well as After adding the detection antibody, a second incubation is performed, followed by the addition of a colorimetric solution to initiate the reaction. The electrochemical impedance is then measured to obtain the electrochemical impedance value.

11. The antigen detection method according to claim 10, characterized in that, The method has one or more of the following conditions: (1) The conditions for the first incubation treatment include: a temperature of 36.8℃~37.2℃ and a time of 10min~20min; (2) The conditions for the second incubation treatment include: a temperature of 36.8℃~37.2℃ and a time of 10min~20min; (3) The chromogenic solution includes a substrate of HRP enzyme; and, (4) The reaction time for adding the colorimetric solution is 30s to 300s.

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