Electrochemical immunosensor as well as preparation method and application thereof

Abstract

The invention belongs to the field of electrochemical immunosensors and the field of protein detection, and particularly relates to an electrochemical immunosensor and a preparation method and application thereof. According to the electrochemical immunosensor provided by the invention, the surface of the electrode is modified by using the gold nanoparticle in-situ modified carbon nanotube, so that not only is the electron transmission efficiency of the electrode effectively improved, but also the effective working area of the surface of the sensor can be increased, and a larger area is provided for subsequent antibody fixation; further, a layer of modified film is formed through self-assembly of Au-S bonds on the surfaces of the gold nanoparticles, so that the surface of the electrode is rich in carboxyl, the carboxyl modification quantity in unit area is effectively increased, the antibodies can be orderly arranged, and the fixing efficiency of the antibodies is maximized. When the electrochemical immunosensor is used for detection, detection signals can be effectively improved, the signal-to-noise ratio is increased, and the detection limit is reduced.

Description

Technical Field

[0001] The present application belongs to the field of electrochemical immunosensors and protein detection, and specifically relates to an electrochemical immunosensor and a preparation method and application thereof. Background Art

[0002] Currently in the medical field, common tumor markers include carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), carbohydrate antigen 125 (CA125) and carbohydrate antigen 199 (CA199). Among them, carcinoembryonic antigen (CEA) is a non-specific tumor marker, and its normal level in human blood is 0μg / L~5μg / L. When it is higher than 20μg / L, it reflects the possibility of cancer in the body, such as colon cancer, pancreatic cancer, breast cancer and gastric cancer. Therefore, the detection of cancer markers can be used as an important means of early cancer screening, and can also be used to monitor tumor recurrence and metastasis.

[0003] Currently, the means of screening CEA are mainly through immunological methods, which use highly specific antigen-antibody affinity reactions to identify trace amounts of antigens in complex body fluids, and then use different signal amplification methods to detect sufficiently high signals. These methods include enzyme-linked immunosorbent assay, chemiluminescence, etc. However, these methods rely on large-scale precision instruments, have complex detection processes and high costs, which limits their application.

[0004] In addition, in the early stages of cancer development, the range of changes in CEA levels in the body is not large, so highly specific and sensitive methods are needed to detect such subtle changes. However, in the traditional technology based on electrochemical sensors, there are still problems with low signal-to-noise ratio and high detection line when detecting CEA in the early stage. Summary of the invention

[0005] Based on this, 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.

[0006] On one hand, the present application provides an electrochemical immunosensor, comprising an electrode substrate, and carbon nanotubes, gold nanoparticles and capture antibodies are sequentially fixed on the surface of the electrode substrate.

[0007] The gold nanoparticles are fixed on the carbon nanotubes.

[0008] The gold nanoparticles are modified with a carboxyl group at the end through an Au-S bond, and the carboxyl group is coupled to the capture antibody through the formation of a peptide bond.

[0009] In one embodiment, the capture antibody comprises one or more of CEA antibody, AFP antibody, CA125 antibody and CA199 antibody.

[0010] In one embodiment, the electrode substrate includes one or more of a screen-printed electrode, a glassy carbon electrode, and a magnetron sputtering electrode.

[0011] Another aspect of the present application provides a method for preparing an electrochemical immunosensor, comprising:

[0012] The carbon nanotubes are fixed on an electrode substrate to prepare a first carbon electrode.

[0013] The first carbon electrode is modified with gold nanoparticles to prepare a second carbon electrode.

[0014] The second carbon electrode is carboxylated, wherein the carboxylation modification is to modify the gold nanoparticles with a carboxyl group at the end by forming an Au-S bond, so as to prepare a third carbon electrode.

[0015] The carboxyl groups on the third carbon electrode are activated, and then the capture antibody is coupled with the activated carboxyl groups to form a peptide bond to prepare an electrochemical immunosensor.

[0016] In one embodiment, preparing the first carbon electrode comprises:

[0017] A carbon nanotube dispersion is prepared, and the carbon nanotube dispersion is coated on the surface of the electrode substrate and dried to prepare a first carbon electrode.

[0018] In one embodiment, the concentration of carbon nanoparticles in the carbon nanotube dispersion is 0.01 wt % to 0 wt %.

[0019] In one embodiment, the drying process includes drying with nitrogen gas.

[0020] In one embodiment, the drying time is 1 h to 2 h.

[0021] In one embodiment, preparing the second carbon electrode comprises: mixing a chloroauric acid solution with the first electrode, and preparing the second carbon electrode by a chemical deposition method.

[0022] In one embodiment, the concentration of the chloroauric acid solution is 0.01wt%~0.5wt%.

[0023] In one embodiment, the conditions of the chemical deposition method include: a voltage of -0.8V to -0.1V, and a time of 20s to 300s.

[0024] In one embodiment, preparing the third carbon electrode comprises: mixing the second carbon electrode with a mixed solution containing a thiol compound and a carboxyl compound, reacting the mixture, and then drying the mixture.

[0025] In one embodiment, the mixed solution containing mercapto compounds and carboxyl compounds includes one or more of mercaptosuccinic acid, 6-(mercaptomethyl)-dodecanedioic acid and 1,3,5-pentanetricarboxylic acid, 3-(2-mercaptoethyl).

[0026] In one embodiment, the concentration of the mercaptosuccinic acid in the mixed solution is 5 mM to 200 mM.

[0027] In one embodiment, the concentration of 6-(mercaptomethyl)-dodecanedioic acid in the mixed solution is 5 mM to 200 mM.

[0028] In one embodiment, the concentration of 1,3,5-pentanetricarboxylic acid in the mixed solution is 5 mM to 200 mM.

[0029] In one embodiment, the concentration of the 3-(2-mercaptoethyl) is 5 mM to 200 mM.

[0030] In one embodiment, the reaction conditions include: temperature of 25°C to 30°C, and time of 12h to 24h.

[0031] In one embodiment, the drying process includes: using nitrogen for drying; optionally, the drying process lasts for 1 hour to 2 hours.

[0032] In one embodiment, the activation treatment includes: activating the third carbon electrode by dripping an activation solution.

[0033] The activation solution includes one or more of 2-(N-morpholine)ethanesulfonic acid, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0034] In one embodiment, the concentration of the 2-(N-morpholine)ethanesulfonic acid in the activation solution is 2 mM to 40 mM.

[0035] In one embodiment, the concentration of N-hydroxysuccinimide is 5 mg / mL to 30 mg / mL.

[0036] In one embodiment, the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 5 mg / mL to 30 mg / mL.

[0037] In one embodiment, the activation treatment time is 0.2h~1h.

[0038] On the other hand, the present application provides an antigen detection method, which uses the above-mentioned electrochemical immunosensor for detection.

[0039] In one embodiment, the method includes: providing a detection antibody; the detection antibody is a conjugate of gold nanoparticles labeled with HRP enzyme and an antibody; and dropping the antigen to be detected onto the surface of the electrochemical immunosensor for a first incubation treatment.

[0040] After adding the detection antibody, a second incubation is performed, a color developing solution is added dropwise for reaction, and the electrochemical impedance is detected to obtain an electrochemical impedance value.

[0041] In one embodiment, the conditions of the first incubation treatment include: a temperature of 36.8°C to 37.2°C and a time of 10 min to 20 min.

[0042] In one embodiment, the conditions of the second incubation treatment include: a temperature of 36.8°C to 37.2°C and a time of 10 min to 20 min.

[0043] In one embodiment, the color developing solution is a substrate of HRP enzyme.

[0044] In one embodiment, the reaction time of adding the color developing solution is 30s to 300s.

[0045] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0047] Figure 1 The specific preparation process and reaction process of the electrochemical immunosensor for this application;

[0048] Figure 2 The impedance test results of the electrodes at different modification stages of this application are shown;

[0049] Figure 3 The time-current curve test results of the electrochemical immunosensor of this application;

[0050] Figure 4 The linear range and detection limit results of the electrochemical immunosensor in measuring CEA antigen in this application. DETAILED DESCRIPTION

[0051] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0052] 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 this application belongs.

[0053] the term

[0054] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0055] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include 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 three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0056] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0057] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0058] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0059] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0060] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0061] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0062] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0063] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0064] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0065] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0066] The term "CEA (carcinoembryonic antigen)": Carcinoembryonic antigen, first isolated from embryonic and tumor tissues, is a broad-spectrum tumor marker.

[0067] The term "AuNP (Au nanoparticles)" refers to gold nanoparticles, a kind of precious metal nanomaterial with a particle size of about 10-100nm, which has nanomaterial properties such as good conductivity and high specific surface area.

[0068] The term "CNTs (carbon nanotubes)" refers to carbon nanotubes, a carbon-based material with different diameters (10nm ~200nm) and lengths (1μm ~10μm), which have nanomaterial properties such as good conductivity and high specific surface area.

[0069] Electrochemical biosensors are sensors that fix biological recognition components (such as antigens, antibodies, enzymes, nucleic acids, etc.) on the surface of the sensor, which recognizes the target substance and causes a series of electrochemical reactions to change the electrical signal. In this system, the sensitivity and efficiency of the sensor can be further improved by adding electroactive substances, redox probes, nanomaterials, etc.

[0070] Nanomaterials and enzyme-catalyzed substrate amplification strategies in electrochemical sensing. The enzyme-catalyzed substrate method amplifies the recognition of antigens and antibodies into more easily detectable changes in electrical signals generated by electroactive products; nanomaterials can increase the fixed amount of antibodies by increasing the specific surface area of ​​the sensor, and can also enrich more enzymes through nanomaterials to further amplify the detection signal.

[0071] Enzyme-linked immunosorbent assay: It is a method of detecting target molecules by using the interaction between antigen and antibody. Antigen or antibody is coated on a polystyrene plate, and after binding to the target molecule, the detection antibody is directly or indirectly coupled to the enzyme, and the amount of the target molecule is converted into the amount of enzyme and the amount of enzyme-catalyzed substrate in proportion. After a color reaction is generated, the target component is quantitatively analyzed by colorimetry.

[0072] Chemiluminescent immunoassay: It is a method of quantitatively detecting target proteins based on antigen-antibody binding. When the target protein forms an antigen-antibody complex with the antibody labeled with a tracer enzyme, the tracer enzyme catalyzes a chemical reaction of the luminescent substrate to produce luminescence, and the luminescence is detected by an optical instrument to correspond to the concentration of the target component.

[0073] On one hand, the present application provides an electrochemical immunosensor, comprising an electrode substrate, and carbon nanotubes, gold nanoparticles and capture antibodies are sequentially fixed on the surface of the electrode substrate.

[0074] Among them, gold nanoparticles are fixed on carbon nanotubes by chemical deposition.

[0075] The gold nanoparticles are modified with a carboxyl group at the end through an Au-S bond, and the carboxyl group is coupled to the capture antibody through the formation of a peptide bond.

[0076] The present application fixes carbon nanotubes on the surface of the electrode, which not only increases the effective working area of ​​the electrode, but also further improves the conductivity of the electrode, effectively improving the efficiency of electron conduction between the electroactive substance and the electrode through the electrochemical reaction.

[0077] Then, gold nanoparticles are grown on the surface of carbon nanotubes by electrochemical deposition. This in-situ growth method can increase the connectivity between gold nanoparticles and carbon nanotubes. Gold has a very high electron transfer efficiency, and the modification of gold nanoparticles can further improve the performance of the electrode. In addition, gold nanoparticles have a large specific surface area, which further increases the antibody binding sites on the electrode and effectively increases the amount of antibody fixation. Finally, the modification of gold nanoparticles allows the electrode surface to be further modified through Au-S bonds.

[0078] In a specific example, the capture antibody includes one or more of one or more of CEA antibody, AFP antibody, CA125 antibody, and CA199 antibody;

[0079] Optionally, the electrode substrate includes, but is not limited to, one or more of a screen-printed electrode, a glassy carbon electrode, and a magnetron sputtering electrode.

[0080] This application uses screen-printed electrodes as a substrate, and further electrode modification is performed on this basis to produce an electrochemical immunosensor. Screen-printed electrodes have low costs, and the sensors produced are disposable, which can reduce problems such as electrode contamination and surface passivation caused by repeated use of sensors. Precise customization and batch production have good repeatability and stability, and can effectively reduce batch differences.

[0081] Another aspect of the present application provides a method for preparing an electrochemical immunosensor, comprising:

[0082] Fixing carbon nanotubes on an electrode substrate to prepare a first carbon electrode;

[0083] Modifying gold nanoparticles on the first carbon electrode by chemical deposition to prepare a second carbon electrode;

[0084] The second carbon electrode is carboxylated, wherein the carboxylation modification is to modify the gold nanoparticles with a carboxyl group at the end by forming an Au-S bond, so as to prepare a third carbon electrode.

[0085] The carboxyl groups on the third carbon electrode are activated, and then the capture antibody is coupled with the activated carboxyl groups through peptide bonds to prepare an electrochemical immunosensor.

[0086] In a specific example, preparing the first carbon electrode includes: preparing a carbon nanotube dispersion, coating the carbon nanotube dispersion on the surface of the electrode substrate, and performing a drying process to prepare the first carbon electrode.

[0087] Optionally, the concentration of the carbon nanotube dispersion is 0.01wt%~1.0wt%; in a specific embodiment, the concentration of the carbon nanotube dispersion is 0.01 wt%, and in a specific embodiment, the concentration of the carbon nanotube dispersion is 1.0wt%; it is understandable 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.

[0088] Optionally, the drying process includes drying with nitrogen gas for 1 hour to 2 hours.

[0089] In one embodiment, preparing the second carbon electrode comprises: mixing a chloroauric acid solution with the first electrode, and preparing the second carbon electrode by chemical deposition.

[0090] Optionally, the concentration of the chloroauric acid solution is 0.01 wt%~0.5 wt%; in a specific embodiment, the concentration of the chloroauric acid solution is 0.01 wt%; in a specific embodiment, the concentration of the chloroauric acid solution is 0.5 wt%; it is understandable 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.

[0091] Optionally, the conditions of the chemical deposition method include: a voltage of -0.8V to 0.2V, and a time of 20s to 300s. In a 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, -0.2V, and other values.

[0092] In one embodiment, preparing the third carbon electrode comprises: mixing the second carbon electrode with a mixed solution containing a thiol compound and a carboxyl compound, reacting the mixture, and then drying the mixture.

[0093] Optionally, the mixed solution containing mercapto compounds and carboxyl compounds includes mercaptosuccinic acid, 6-(mercaptomethyl)-dodecanedioic acid and 1,3,5-pentanetricarboxylic acid, 3-(2-mercaptoethyl).

[0094] In a specific example, the concentration of mercaptosuccinic acid in the mixed solution is 5 mM to 200 mM.

[0095] For example, 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM or 200mM.

[0096] Optionally, the concentrations of 6-(mercaptomethyl)-dodecanedioic acid and in the mixed solution are 5 mM to 200 mM, respectively, such as 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.

[0097] Optionally, the concentration of 1,3,5-pentanetricarboxylic acid in the mixed solution 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.

[0098] The carboxyl groups modified on the electrode surface were activated by N-hydroxysuccinimide solution and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, so that they could undergo condensation reaction with the amino groups of the antibody, coupling the antibody to the electrode, and finally blocking the excess active sites to prevent nonspecific adsorption.

[0099] 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.

[0100] In a specific example, the reaction conditions include: temperature of 25°C ~ 30°C, and time of 12h ~ 24h; for example, the temperature is 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, and the time is 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h and other values.

[0101] In a specific example, the drying process includes: using nitrogen to perform drying process, and the process time is 1 hour to 2 hours.

[0102] Optionally, the activation treatment comprises: performing an activation treatment on the third carbon electrode by dripping an activation solution;

[0103] The activating solution includes 2-(N-morpholine)ethanesulfonic acid, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;

[0104] Optionally, the concentration of 2-(N-morpholine)ethanesulfonic acid is 2mM~40mM; in a specific embodiment, the concentration of 2-(N-morpholine)ethanesulfonic acid is 2mM, and in a specific embodiment, the concentration of 2-(N-morpholine)ethanesulfonic acid is 40mM; it is understandable that the concentration of 2-(N-morpholine)ethanesulfonic acid is 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM or other values.

[0105] Optionally, the concentration of N-hydroxysuccinimide is 5 mg / mL to 30 mg / mL; in a specific embodiment, the concentration of N-hydroxysuccinimide is 5 mg / mL, and in a specific embodiment, the concentration of N-hydroxysuccinimide is 30 mg / mL; it is understandable that the concentration of N-hydroxysuccinimide is 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL or other values.

[0106] Optionally, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 5 mg / mL to 30 mg / mL. In a specific embodiment, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 5 mg / mL, and in a 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.

[0107] Optionally, the activation treatment time is 0.2h~1h. For example, the time is 0.2h, 0.3h, 0.4h, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h.

[0108] In a specific example, the capture antibody is one or more of CEA antibody, AFP antibody, CA125 antibody and CA199 antibody.

[0109] Optionally, the concentration of CEA antibody is 5 μg / mL to 125 μg / mL. In a specific embodiment, the concentration of CEA antibody is 5 μg / mL, and in a specific embodiment, the concentration of CEA antibody is 125 μg / mL; it is understood that the concentration of CEA antibody is 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL or other values.

[0110] On the other hand, the present application provides an antigen detection method, which uses the above-mentioned electrochemical immunosensor for detection.

[0111] In a specific example, the method includes: providing a detection antibody; the detection antibody is a conjugate of gold nanoparticles labeled with HRP enzyme and an antibody.

[0112] The antigen to be tested is added dropwise onto the surface of the electrochemical immunosensor for a first incubation treatment.

[0113] After adding the detection antibody, a second incubation is performed, a color developing solution is added dropwise for reaction, and the electrochemical impedance is detected to obtain an electrochemical impedance value.

[0114] After the immunosensor binds to the antigen, it is necessary to add the detection antibody labeled with HRP enzyme for characterization. When the detection antibody binds to the electrode through the antigen-antibody reaction, the HRP content on the electrode surface will increase. After the substrate is added, HRP catalyzes the substrate to generate an electroactive product, and a redox reaction occurs at a specific voltage, resulting in a change in current.

[0115] In terms of detecting antibody labeling, gold nanoparticles are used as carriers to load as much HRP enzyme as possible, and then labeled on the antibody. Compared with directly labeling HRP on the antibody, loading a large amount of HRP on nanoparticles can effectively increase the HRP loading amount per unit concentration of antibody, thereby increasing the amount of product, and ultimately leading to a larger value of current change, thereby effectively improving the sensitivity of the sensor.

[0116] In a specific example, the conditions of the first incubation treatment include: a temperature of 36°C to 38°C, and a time of 10 min to 20 min. For example, the temperature is 36°C, 37°C or 38°C, and the time is 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min.

[0117] In a specific example, the conditions of the second incubation treatment include: a temperature of 36°C to 38°C, and a time of 10 min to 20 min. For example, the temperature is 36°C, 37°C or 38°C, and the time is 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min.

[0118] In one specific example, the developing solution includes a substrate for the HRP enzyme.

[0119] In a specific example, the reaction time of adding the color developing solution is 30s to 300s, for example, 30s, 50s, 70s, 90s, 110s, 130s, 150s, 170s, 190s, 210s, 230s, 250s, 270s, 290s, and 300s.

[0120] The present application provides an electrochemical immunosensor, which not only effectively improves the electron transfer efficiency of the electrode, but also increases the effective working area of ​​the sensor surface by modifying the electrode surface with carbon nanotubes modified with gold nanoparticles in situ, providing a larger area for subsequent antibody fixation, and further self-assembles a layer of modified film on the surface of the gold nanoparticles through Au-S bonds, so that the electrode surface is rich in carboxyl groups, thereby effectively increasing the number of carboxyl modifications per unit area, enabling the orderly arrangement of antibodies, thereby maximizing the antibody fixation efficiency. When using the electrochemical immunosensor of the present application for detection, the detection signal can be effectively improved, the signal-to-noise ratio can be increased, and the detection limit can be reduced.

[0121] In addition, when the present application is used for testing, the sample does not need to be processed, the detection process is few and the time is short, so the detection can be carried out more efficiently and the sensitivity can be improved; subsequently, the microfluidic chip can be designed to make it automated, thereby reducing manual operations to a great extent, achieving high-throughput testing, and facilitating the development towards POCT.

[0122] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0123] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0124] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean 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 the present application.

[0125] Example 1

[0126] This embodiment provides a method for preparing an electrochemical immunosensor. The specific preparation process and reaction process are as follows: Figure 1 As shown, specifically including:

[0127] 1. Preparation of CNTs dispersion

[0128] Chitosan was weighed and added to a 0.05 M sodium acetate buffer solution with a pH of 5.0, and the solution was stirred thoroughly to obtain a 2.5 wt% chitosan solution. Carbon nanotube powder was then added thereto and ultrasonically dispersed at 300 W for 3 h to obtain a black CNTs dispersion solution with a concentration of 0.5 wt%.

[0129] 2. Modification of CNTs (first electrode)

[0130] Screen-printed electrodes were selected for making sensors, which used carbon electrodes as working electrodes and counter electrodes, and Ag / AgCl electrodes as reference electrodes. CNTs dispersion droplets were applied to the clean working electrode surface and dried under an infrared lamp. After gently rinsing with PBS solution, the electrode surface was blown dry with nitrogen to prepare a CNTs-modified carbon electrode (C-CNTs electrode).

[0131] 3. Modification of AuNPs (second electrode)

[0132] A 0.25% chloroauric acid solution was added to the electrode area of ​​the SPCE, and AuNPs were deposited on the surface of the working electrode C-CNTs electrode at a voltage of 0.2 V using the chronoamperometry method. The deposition time was 250 s to obtain a carbon electrode doubly modified with AuNPs and CNTs (C-CNTs / AuNP electrode).

[0133] 4. Modification of carboxyl group (third electrode)

[0134] The screen-printed electrode was treated with 150 mM mercaptosuccinic acid and reacted at room temperature overnight to spontaneously form a carboxyl-rich self-assembled film on the surface of the gold nanoparticles through Au-S bonds. The electrode was then gently washed with PBS solution and the electrode surface was blown dry with nitrogen gas.

[0135] 5. Activation of carboxyl groups

[0136] After mixing equal volumes of 25 mM 2-(N-morpholine)ethanesulfonic acid solution, 20 mg / mL N-hydroxysuccinimide solution, and 20 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution, 25 μL of the mixture was dropped onto the C-CNTs / AuNP electrode and activated at room temperature for 1 hour. After it was semi-dried, 25 μL of 85 μg / mL CEA antibody was dropped onto the surface of the working electrode, and the electrode was placed in a wet box at 37°C for 2 hours to react the amino groups on the antibody with the activated carboxyl groups on the self-assembled membrane to couple the antibody. After that, the electrode was cleaned with PBST to remove the unstably bound CEA antibody, and the C-CNTs / AuNP-Ab electrode was obtained.

[0137] 6. Sealing of electrodes

[0138] 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 cleaned and dried for later use.

[0139] 7. Preparation of gold nanoparticles

[0140] Gold nanoparticles were prepared using the reduction method. A 0.25 wt % chloroauric acid solution was heated to 100 ° C. An appropriate amount of trisodium citrate was added during stirring to make the final concentration in the mixed solution 2.5 wt %. The reaction was continued to generate a red solution. The solution was heated and boiled at 100 ° C for 25 min. After cooling to room temperature, deionized water was added to prepare a 1% gold nanoparticle solution.

[0141] 8. Gold nanoparticles labeled antibodies and HRP enzyme

[0142] Use K 2 CO 3 The pH of 5 mL of gold nanoparticle solution was adjusted to 7, and 0.25 mg of CEA monoclonal antibody and 1 mg of HRP enzyme were added respectively. After being fully mixed, they were incubated at room temperature for 3 h, and then 20% wt BSA solution was added to block the gold nanoparticle surface. The solution was centrifuged at 12,000 rpm for 20 min and washed three times to obtain HRP-rich gold nanoparticle-labeled CEA antibody (AuNP / HRP-Ab).

[0143] Example 2

[0144] This embodiment provides a method for preparing an electrochemical immunosensor, comprising:

[0145] 1. Preparation of CNTs dispersion

[0146] Chitosan was weighed and added to a sodium acetate buffer solution with a concentration of 0.01 M and a pH of 4.0, and then stirred thoroughly to obtain a chitosan solution with a concentration of 0.1 wt%. Carbon nanotube powder was then added thereto and ultrasonically dispersed at a power of 300 W for 2 h to obtain a black CNTs dispersion with a concentration of 0.01 wt%.

[0147] 2. Modification of CNTs (first electrode)

[0148] Screen-printed electrodes were selected for making sensors, which used carbon electrodes as working electrodes and counter electrodes, and Ag / AgCl electrodes as reference electrodes. CNTs dispersion droplets were applied to the clean working electrode surface and dried under an infrared lamp. After gently rinsing with PBS solution, the electrode surface was blown dry with nitrogen to prepare a CNTs-modified carbon electrode (C-CNTs electrode).

[0149] 3. Modification of AuNPs (second electrode)

[0150] 0.01% chloroauric acid solution was added to the electrode area of ​​​​SPCE, and AuNPs were deposited on the surface of the working electrode C-CNTs electrode at a voltage of -0.8 V using the chronoamperometry method. The deposition time was 20 s to obtain a carbon electrode doubly modified with AuNPs and CNTs (C-CNTs / AuNP electrode).

[0151] 4. Modification of carboxyl group (third electrode)

[0152] The screen-printed electrode was treated with an aqueous solution of 6-(mercaptomethyl)-dodecanedioic acid at a concentration of 150 mM. After reacting at room temperature overnight, a carboxyl-rich self-assembled film was spontaneously formed on the surface of the gold nanoparticles through Au-S bonds. The electrode was then gently washed with PBS solution and the electrode surface was blown dry with nitrogen gas.

[0153] 5. Activation of carboxyl groups

[0154] After mixing equal volumes of 2-(N-morpholine)ethanesulfonic acid solution with a concentration of 2mM, N-hydroxysuccinimide solution with a concentration of 5 mg / mL, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution with a concentration of 20 mg / mL, 5μL of the mixture was dropped onto the C-CNTs / AuNP electrode and activated at room temperature for 0.2h. After it was semi-dried, 5μL of CEA antibody with a concentration of 5μg / mL was dropped onto the surface of the working electrode, and the electrode was placed in a wet box at 37℃ for 1h to react so that the amino groups on the antibody and the activated carboxyl groups on the self-assembled membranes reacted to couple the antibody. After that, the electrode was cleaned with PBST to remove the unstably bound CEA antibody, and the C-CNTs / AuNP-Ab electrode was obtained.

[0155] 6. Sealing of electrodes

[0156] The C-CNTs / AuNP-Ab electrode was immersed in a solution containing 5 wt% CAS and sealed at 37°C for 1 h. The electrode was cleaned and dried for later use.

[0157] 7. Preparation of gold nanoparticles

[0158] Gold nanoparticles were prepared using the reduction method. A 0.01 wt% chloroauric acid solution was heated to 100°C. An appropriate amount of sodium citrate solution was added during stirring to a final concentration of 1 wt% in the mixed solution. The solution was allowed to react continuously to generate a red solution. The solution was heated and boiled at 100°C for 5 min. After cooling to room temperature, deionized water was added to prepare a 1% gold nanoparticle solution.

[0159] 8. Gold nanoparticles labeled antibodies and HRP enzyme

[0160] Use K 2 CO 3The pH of 5 mL of gold nanoparticle solution was adjusted to 6, and 0.02 mg of CEA monoclonal antibody and 0.1 mg of HRP enzyme were added respectively. After being fully mixed, they were incubated at room temperature for 2 h, and then 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).

[0161] Example 3

[0162] This embodiment provides a method for preparing an electrochemical immunosensor, comprising:

[0163] 1. Preparation of CNTs dispersion

[0164] Chitosan was weighed and added to a 0.1 M sodium acetate buffer solution with a pH of 6.0, and the solution was stirred thoroughly to obtain a chitosan solution with a concentration of 5.0 wt%. Carbon nanotube powder was then added thereto and ultrasonically dispersed at a power of 300 W for 4 h to obtain a black CNTs dispersion with a concentration of 1.0 wt%.

[0165] 2. Modification of CNTs (first electrode)

[0166] Screen-printed electrodes were selected for making sensors, which used carbon electrodes as working electrodes and counter electrodes, and Ag / AgCl electrodes as reference electrodes. CNTs dispersion droplets were applied to the clean working electrode surface and dried under an infrared lamp. After gently rinsing with PBS solution, the electrode surface was blown dry with nitrogen to prepare a CNTs-modified carbon electrode (C-CNTs electrode).

[0167] 3. Modification of AuNPs (second electrode)

[0168] A 0.5% chloroauric acid solution was added to the electrode area of ​​the SPCE, and AuNPs were deposited on the surface of the working electrode C-CNTs electrode at a voltage of 0.2 V using the chronoamperometry method. The deposition time was 300 s to obtain a carbon electrode doubly modified with AuNPs and CNTs (C-CNTs / AuNP electrode).

[0169] 4. Modification of carboxyl group (third electrode)

[0170] The screen-printed electrode was treated with a 200 mM aqueous solution of 3-(2-mercaptoethyl) and reacted at room temperature overnight to spontaneously form a carboxyl-rich self-assembled film on the surface of the gold nanoparticles through Au-S bonds. The electrode was then gently washed with PBS solution and the electrode surface was blown dry with nitrogen gas.

[0171] 5. Activation of carboxyl groups

[0172] After mixing equal volumes of 40 mM 2-(N-morpholine)ethanesulfonic acid solution, 30 mg / mL N-hydroxysuccinimide solution and 30 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution, 50 μL of the mixture was added to the C-CNTs / AuNP electrode and activated at room temperature for 1 hour. After it was half dry, 50 μL of 125 μg / mL CEA antibody was added to the surface of the working electrode and placed in a wet box at 37°C for 4 hours to react the amino groups on the antibody with the activated carboxyl groups on the self-assembled membrane to couple the antibody. After that, the electrode was cleaned with PBST to remove the unstable CEA antibody and obtain the C-CNTs / AuNP-Ab electrode.

[0173] 6. Closure of electrodes

[0174] The C-CNTs / AuNP-Ab electrode was immersed in a solution containing 10 wt% PEG and sealed at 37°C for 2 h. The electrode was cleaned and dried for later use.

[0175] 7. Preparation of gold nanoparticles

[0176] Gold nanoparticles were prepared using the reduction method. A 0.5 wt% chloroauric acid solution was heated to 100°C. An appropriate amount of tannic acid solution was added during stirring to a final concentration of 5 wt% in the mixed solution. The reaction was continued to generate a red solution. The solution was heated and boiled at 100°C for 30 min. After cooling to room temperature, deionized water was added to prepare a 1% gold nanoparticle solution.

[0177] 8. Gold nanoparticles labeled antibodies and HRP enzyme

[0178] Use K 2 CO 3 The pH of 5 mL of gold nanoparticle solution was adjusted to 6, and 0.5 mg of CEA monoclonal antibody and 2 mg of HRP enzyme were added respectively. After being fully mixed, the mixture was incubated at room temperature for 6 h, and then 20% wt BSA solution was added to block the surface of the gold nanoparticles. The mixture was centrifuged at 15,000 rpm for 30 min and washed three times to obtain HRP-rich gold nanoparticle-labeled CEA antibody (AuNP / HRP-Ab).

[0179] CEA antigen test results test:

[0180] Gradient concentrations of CEA antigen were prepared using antibody diluent and added to the prepared C-CNTs / AuNP-Ab electrode surface. The mixture was incubated at 37°C for 15 min and washed with PBST. AuNP / HRP-Ab solution was added and incubated at 37°C for 15 min. TMB solution was added after washing with PBST. After reacting for 300 s, the current was tested at a voltage of 0.1 V using the chronoamperometry method.

[0181] The electrodes at different modification stages were subjected to impedance tests. A solution containing 5 mM potassium ferrocyanide and 5 mM potassium chloride was used as the electrolyte solution. The impedance spectrum test was performed at an open circuit voltage with an amplitude modulation of 0.01 V. The test results are as follows: Figure 2 shown.

[0182] The results showed that after the electrode was modified with CNTs and AuNPs, the impedance of the electrode was significantly reduced, because both had good conductivity and increased the surface area of ​​the electrode, which was beneficial to the electrochemical reaction between potassium ferrocyanide and the electrode surface; after the electrode was modified and activated by the linker and coupled with the antibody, the impedance increased significantly, which was due to the increase of non-conductive biological components on the electrode surface, which hindered the electron transfer between potassium ferrocyanide and the electrode, indicating that the present application can effectively fix a large amount of antibody protein.

[0183] In addition, the nanomaterial modification on the electrode increases the binding area of ​​the antibody, and the polycarboxyl compounds modified on the nanomaterial increase the number of carboxyl groups per unit area on the electrode, further promoting the binding of more antibodies. After blocking, the impedance value increases further, and the surface of the surface electrode is covered with more non-conductive components, indicating that many non-specific sites on the electrode are blocked.

[0184] Detection limit test:

[0185] 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 curve was as follows: Figure 3 As shown, the current value at 60 seconds is taken for statistics, and a curve of the current signal with respect to the antigen concentration is drawn.

[0186] from Figure 3 It can be seen that the current value increases with the increase of CEA antigen concentration, because the number of antigens bound to the capture antibody increases, and more detection antibodies are bound to the electrode surface. The detection antibody is connected with HRP-rich gold nanoparticles. After adding the substrate, more HRP plays a catalytic role to generate electroactive products, which will produce a higher current signal under the same voltage.

[0187] like Figure 4 The results showed that when the electrochemical immunosensor measured CEA antigen, the linear range was 0.1ng / mL~20ng / mL, R2=0.997, and the detection limit was 0.04ng / mL.

[0188] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description 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 are sequentially fixed on the surface of the electrode substrate; The gold nanoparticles are fixed on the carbon nanotubes; The gold nanoparticles are modified with a carboxyl group at the end through an Au-S bond, and the carboxyl group is coupled to the capture antibody through the formation of a peptide bond.

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

3. A method for preparing an electrochemical immunosensor, characterized in that: include: Fixing the carbon nanotubes on an electrode substrate to prepare a first carbon electrode; Modifying gold nanoparticles on the first carbon electrode to prepare a second carbon electrode; Carboxylation modification is performed on the second carbon electrode, wherein the carboxylation modification is to modify the gold nanoparticles with a carboxyl group at the end by forming an Au-S bond, so as to prepare a third carbon electrode; and The carboxyl groups on the third carbon electrode are activated, and then the capture antibody is coupled with the activated carboxyl groups to form a peptide bond to prepare an electrochemical immunosensor.

4. The method for preparing the electrochemical immunosensor according to claim 3, characterized in that: Preparing a first carbon electrode comprises: preparing a carbon nanotube dispersion, coating the carbon nanotube dispersion on the surface of the electrode substrate, and performing a drying process 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; Optionally, the drying time is 1 h to 2 h.

5. The method for preparing the electrochemical immunosensor according to claim 3, characterized in that: Preparing the second carbon electrode comprises: Mixing the chloroauric acid solution with the first electrode, and preparing a second carbon electrode by chemical deposition; Optionally, the concentration of the chloroauric acid solution is 0.01 wt%~0.5 wt%; Optionally, the conditions of the chemical deposition method include: voltage of -0.8 V to -0.1 V, and 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 comprises: The second carbon electrode is mixed with a mixed solution containing a thiol compound and a carboxyl compound to react, and then dried; Optionally, the mixed solution containing mercapto compounds and carboxyl compounds includes one or more of mercaptosuccinic acid, 6-(mercaptomethyl)-dodecanedioic acid and 1,3,5-pentanetricarboxylic acid, 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 mixed solution is 5 mM to 200 mM; (2) the concentrations of 6-(mercaptomethyl)-dodecanedioic acid and in the mixed solution are 5 mM to 200 mM respectively; (3) The concentration of 1,3,5-pentanetricarboxylic acid in the mixed solution is 5 mM to 200 mM; (4) The concentration of the 3-(2-mercaptoethyl) is 5 mM to 200 mM; (5) The reaction conditions include: temperature of 25°C to 30°C and time of 12h to 24h; and, (6) Drying treatment includes: drying treatment using nitrogen; Optionally, the drying time is 1 h to 2 h.

8. The method for preparing the electrochemical immunosensor according to claim 3, characterized in that: Activation treatment includes: Activating the third carbon electrode by adding an activation solution; The activation solution includes one or more of 2-(N-morpholine)ethanesulfonic acid, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; Optionally, the concentration of the 2-(N-morpholine)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~1h.

9. An antigen detection method, characterized in that: The detection is performed using the electrochemical immunosensor according to any one of claims 1 to 2.

10. The antigen detection method according to claim 9, characterized in that: include: Providing a detection antibody; the detection antibody is a conjugate of gold nanoparticles and antibodies labeled with HRP enzyme; Adding the antigen to be tested dropwise onto the surface of the electrochemical immunosensor for a first incubation treatment; as well as After adding the detection antibody, a second incubation is performed, a color developing solution is added dropwise for reaction, and the electrochemical impedance is detected to obtain an 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 of the first incubation treatment include: a temperature of 36.8°C to 37.2°C and a time of 10 min to 20 min; (2) The conditions of the second incubation treatment include: a temperature of 36.8°C to 37.2°C and a time of 10 min to 20 min; (3) The color developing solution includes a substrate for HRP enzyme; and (4) The reaction time of adding the color developing solution is 30s~300s.

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