A detection method for NR2F1 antigen and its reaction pool

Abstract

The invention discloses a method for detecting NR2F1 antigen and a reaction pool thereof, belongs to the field of biotechnology, solves the problems of high detection cost of existing detection methods, and comprises the following steps: step A: preparing a second NR2F1 antibody solution modified by glucose oxidase; step B: preparing an electrode paper sheet of a working electrode modified by a first NR2F1 antibody; step C: inserting the electrode paper sheet in step B into the reaction pool, dripping NR2F1 antigen extract into the reaction pool, dripping the second NR2F1 antibody solution modified by glucose oxidase in step A, reacting for 1 to 3 minutes, dripping glucose solution, reacting for 20 to 40 seconds, and detecting the current; step D: dripping NR2F1 antigen extracts of different concentrations and repeating step C, and making a standard curve according to the relationship between the antigen concentration and the current value; step E: taking a sample to be tested for detection, and obtaining the NR2F1 antigen concentration value in the sample to be tested. The invention has the advantages of portable detection and low cost.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a detection method for NR2F1 antigen and a reaction pool thereof. Background Art

[0002] The NR2F1 level in tumor cells determines whether the tumor cells are dormant or activated, and can predict whether the metastatic tumor cells in breast cancer patients are lethal. The NR2F1 level in tumor cells in the bone marrow of breast cancer patients can be measured to predict the subsequent risk of death. Androgen deprivation therapy is associated with increased NR2F1 levels. Early androgen deprivation therapy helps reduce the risk of breast cancer recurrence. Therefore, the detection of NR2F1 levels in tumor cells in the bone marrow is of great significance for the treatment of breast cancer. When diagnosing breast cancer, medical staff can detect the NR2F1 antigen concentration as a reference indicator in the early stage.

[0003] However, some existing detection methods have the following disadvantages when detecting NR2F1: first, large equipment is expensive and requires professional operation; second, the test needs to be carried out in a professional laboratory, which is not convenient for the subjects to test; third, it takes a long time and the operation is cumbersome. Therefore, it is necessary to find a simple, fast and timely method to detect NR2F1. Summary of the invention

[0004] The first object of the present invention is to provide a method for detecting NR2F1 antigen in view of the above-mentioned problems existing in the prior art. The second object of the present invention is to provide a reaction pool for implementing the above-mentioned method.

[0005] The first object of the present invention can be achieved by the following technical scheme: A method for detecting NR2F1 antigen comprises the following steps:

[0006] Step A: preparing a glucose oxidase-modified NR2F1 secondary antibody solution;

[0007] Step B: preparing an electrode paper sheet for modifying a working electrode with the NR2F1 primary antibody;

[0008] Step C: insert the electrode paper in step B into the reaction pool, drip NR2F1 antigen extract into the reaction pool, then drip the NR2F1 second antibody solution modified by glucose oxidase in step A, react for 1 to 3 minutes, drip glucose solution, react for 20 to 40 seconds, and detect the current;

[0009] Step D: Repeat step C by adding different concentrations of NR2F1 antigen extracts, and make a standard curve based on the relationship between antigen concentration and current value;

[0010] Step E: Take the sample to be tested for testing to obtain the NR2F1 antigen concentration value in the sample to be tested.

[0011] Preferably, the preparation method of the glucose oxidase modified NR2F1 second antibody solution in step A is: take 10ml 0.2mg / ml nano titanium dioxide solution and add it dropwise to 8ml 0.1mg / ml 3-aminopropyltrimethoxysilane methanol solution to obtain a mixed solution a; mix the mixed solution a with 18ml 0.1mg / ml nano silver sol to form a mixed solution b; add 2μl 5μg / ml NR2F1 second antibody to the mixed solution b to obtain a mixed solution c; mix the mixed solution c with 10ml 0.2mg / ml glucose oxidase to obtain a glucose oxidase modified NR2F1 second antibody solution. First, the nano titanium dioxide is aminated with 3-aminopropyltrimethoxysilane methanol. The amino group has good adsorption performance for nano silver particles, and nano silver can effectively adsorb proteins. Nano titanium dioxide has excellent enzyme adsorption performance, and finally the glucose oxidase modified NR2F1 second antibody solution can be obtained.

[0012] Preferably, in step B: 2 μl of 0.1 mg / ml graphene-perfluorosulfonic acid solution is applied to the carbon electrode of the electrode paper; 1 μl of 0.1 mg / ml cysteine ​​solution is dripped onto the electrode; the nanosilver sol is dripped onto the electrode surface; 2 μl of 5 μg / ml NR2F1 first antibody solution is dripped onto the electrode surface, and finally dried at room temperature to form a working electrode, thereby obtaining an electrode paper with a NR2F1 first antibody modified working electrode. After the graphene-perfluorosulfonic acid solution is applied to the electrode paper, a layer of graphene-perfluorosulfonic acid film can be formed, and after cysteine ​​is dripped onto the carbon electrode, a thiol group is formed to bind to the nanosilver particles to form a nanosilver film, and the nanosilver can well adsorb the NR2F1 first antibody.

[0013] Preferably, in step C, the electrode paper in step B is inserted into the reaction pool, 0.5 ml of NR2F1 antigen extract is dripped into the reaction pool, and then 1 ml of glucose oxidase-modified NR2F1 second antibody solution in step A is dripped, and after reacting for 2 minutes, 2 ml of 0.1 mg / ml glucose solution is dripped, and after reacting for 30 seconds, the current is detected.

[0014] Preferably, in step C, before dripping the glucose solution, the working electrode and the reaction pool of the electrode paper need to be rinsed with deionized water. The deionized water rinses away the excess second antibody to reduce errors.

[0015] Preferably, in step D, the concentrations of the NR2F1 antigen extracts are 5.0×10 -9 mol / L、1.0×10 -8 mol / L、5.0×10 -8mol / L、1.0×10 -7 mol / L、5.0×10 -7 mol / L、1.0×10 -6 mol / L、1.5×10 -6 mol / L、2.0×10 -6 mol / L. According to the relationship between concentration and current value, a standard curve is made, and finally the unknown concentration of NR2F1 can be detected.

[0016] Preferably, in step E, the electrode paper in step B is inserted into the reaction cell, the sample to be tested is added dropwise, and then 1 ml of the glucose oxidase-modified NR2F1 second antibody solution in step A is added dropwise. After reacting for 2 minutes, 2 ml of 0.1 mg / ml glucose solution is added dropwise. After reacting for 30 seconds, the current is detected and brought into the standard curve in step D to calculate the NR2F1 antigen concentration value in the sample to be tested.

[0017] The second object of the present invention can be achieved by the following technical scheme: a reaction pool, the reaction pool is used to implement the above-mentioned NR2F1 antigen detection method, the reaction pool includes a base, the base is penetrated by a cleaning pipe, a reaction chamber is arranged in the cleaning pipe, the top of the reaction chamber is provided with an opening, the top of the base is provided with a groove, the opening is located at the bottom of the groove, the opening is used for one end of the electrode paper to be inserted into the reaction chamber (3), the base is also provided with a liquid inlet channel, the liquid inlet channel is connected to the reaction chamber, the liquid inlet channel is used to facilitate the addition of reaction liquid, the two sides of the reaction chamber are provided with a first one-way valve, when the first one-way valve is opened, the reaction chamber and the cleaning pipe are connected, the cleaning pipe is used for the circulation of water, one end of the cleaning pipe is connected to a water inlet pipe, the other end of the cleaning pipe is connected to a water outlet pipe, the water inlet pipe is used for water entry, the water outlet pipe is used for water discharge, the water inlet pipe is provided with a peristaltic pump, and the peristaltic pump is used to control the flow rate of water. 0.1mg / ml 1ul

[0018] Preferably, the base is made of transparent plastic for easy observation.

[0019] Beneficial effects of the present invention:

[0020] 1. The present invention forms a microcurrent through antibody-antigen-antibody binding, and then calculates the concentration of the antigen by detecting the current based on the electron transfer generated by glucose catalyzed by glucose oxidase. A biosensor electrode paper is used to detect the antigen concentration simply and conveniently. Compared with expensive test kits, this method has low detection cost and is more convenient. In addition, the results of this method are highly reproducible and the time to produce results is short, so it can be used for bedside detection.

[0021] 2. The present invention utilizes the adsorption performance of nanosilver on antibodies without affecting their biological activity. Therefore, it can be made into a working electrode that adsorbs the first antibody of NR2F1. The traditional protein directional immobilization method is to directly adsorb the protein on the surface of the gold electrode, which is easy to fall off, and the flat gold is easy to cause protein denaturation, reduce the activity of the protein, and thus affect the ability of the protein to bind to the antibody. The method of using nanosilver to fix the protein provides an environment similar to the redox protein itself, maintaining the biological activity of the protein.

[0022] 3. The present invention utilizes the adsorption characteristics of amino-modified nano-titanium dioxide on nano-silver particles to adsorb glucose oxidase modified on the NR2F1 second antibody. Nano-grade titanium dioxide, with a diameter of less than 100 nanometers, has the characteristics of antibacterial, self-cleaning, anti-aging properties and good dispersibility.

[0023] 4. The graphene of the present invention has high mechanical strength, good electronic conductivity and stable chemical properties. The screen-printed electrode modified with graphene has the characteristics of strong anti-interference ability, good stability, long service life, easy preparation and operation, fast and sensitive detection, etc. Perfluorosulfonic acid, as a film-forming material and dispersant, can stably fix graphene on the electrode surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a structural schematic diagram of the reaction pool of the present invention;

[0025] Figure 2 It is a schematic diagram of the cross-sectional structure of the reaction tank of the present invention.

[0026] In the figure, 1. base; 2. cleaning pipe; 3. reaction chamber; 4. opening; 5. groove; 6. liquid inlet channel; 7. first one-way valve; 11. water inlet pipe; 12. water outlet pipe; 13. peristaltic pump. DETAILED DESCRIPTION

[0027] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0028] Example 1

[0029] Preparation of glucose oxidase-modified NR2F1 secondary antibody solution

[0030] First, 10 ml of nano titanium dioxide solution (0.2 mg / ml) was added dropwise to 8 ml of 3-aminopropyltrimethoxysilane methanol solution (0.1 mg / ml) to obtain a mixed solution a;

[0031] Then, the mixed solution a was mixed with 18 ml of nanosilver sol (0.1 mg / ml) to form a mixed solution b, and then 2 μl of NR2F1 second antibody (5 μg / ml) was added to the mixed solution b to obtain a mixed solution c, and then the mixed solution c was mixed with 10 ml of glucose oxidase (0.2 mg / ml) to finally obtain a glucose oxidase-modified NR2F1 second antibody solution.

[0032] Preparation of electrode paper for NR2F1 primary antibody modified working electrode

[0033] A 1 mm thick PET plate was used as the substrate. The substrate surface was cleaned with distilled water and acetone, and the silver layer, carbon layer and insulating layer were printed on the substrate in sequence using a screen printing process. A reference electrode, a working electrode and a counter electrode were provided on the substrate of the electrode paper, and the reference electrode and the counter electrode were micro silver / silver chloride electrodes. 2 μl of graphene-perfluorosulfonic acid solution (0.1 mg / ml) was applied to the carbon electrode of the electrode paper, 1 μl of cysteine ​​solution (0.1 mg / ml) was dripped onto the electrode, and then the nanosilver sol was dripped onto the electrode surface, followed by dripping 2 μl of NR2F1 first antibody solution (5 μg / ml) onto the electrode surface, and finally dried at room temperature to form a working electrode, thereby obtaining an electrode paper with NR2F1 first antibody modified working electrode.

[0034] Preparation of standard curve

[0035] Insert the electrode paper of the NR2F1 first antibody modified working electrode into the reaction pool, drip 0.5 ml of NR2F1 antigen extract of different concentrations into the reaction pool, then drip the NR2F1 second antibody solution modified by glucose oxidase in step A, react for 1 to 3 minutes, then drip 2 ml of glucose solution (0.1 mg / ml), react for 20 to 40 seconds, and detect the current; the results are shown in Table 1:

[0036] Concentration of NR2F1 antigen extract Current value <![CDATA[5.0×10 -9 mol / L]]> 0.0260μA <![CDATA[1.0×10 -8 mol / L]]> 0.0265μA <![CDATA[5.0×10 -8 mol / L]]> 0.0282μA <![CDATA[1.0×10 -7 mol / L]]> 0.0310μA <![CDATA[5.0×10 -7 mol / L]]> 0.0451μA <![CDATA[1.0×10 -6 mol / L]]> 0.0669μA <![CDATA[1.5×10 -6 mol / L]]> 0.0870μA <![CDATA[2.0×10 -6 mol / L]]> 0.1101μA

[0037] Results: The standard curve equation is: I = 0.02581 + 0.04150c (I is the test current value, c is the NR2F1 antigen concentration), the detection linear range is 5.0 × 10 -9 mol / L-2.0×10 -6 mol / L, the fitting factor R2 is 0.99897.

[0038] Testing of samples to be tested

[0039] Insert the electrode paper of the NR2F1 first antibody modified working electrode into the reaction pool, add the sample to be tested, and then add 1 ml of the NR2F1 second antibody solution modified with glucose oxidase in step A. After reacting for 2 minutes, add 2 ml of 0.1 mg / ml glucose solution. After reacting for 30 seconds, detect the current and bring it into the standard curve in step D to calculate the NR2F1 antigen concentration value in the sample to be tested.

[0040] Samples to be tested Current value NR2F1 antigen concentration 1 0.03770μA <![CDATA[2.8×10 -7 mol / L]]> 2 0.02917μA <![CDATA[8.1×10 -8 mol / L]]> 3 0.03259μA <![CDATA[1.6×10 -7 mol / L]]>

[0041] The reaction pool in the present invention is used to implement the above-mentioned NR2F1 antigen detection method. The reaction pool includes a base 1, a cleaning pipe 2 is passed through the base 1, a reaction chamber 3 is arranged in the cleaning pipe 2, an opening 4 is arranged on the top of the reaction chamber 3, a groove 5 is opened on the top of the base 1, the opening 4 is located at the bottom of the groove 5, and the opening 4 is used to insert one end of the electrode paper into the reaction chamber 3. The base 1 is also provided with a liquid inlet channel 6, which is connected to the reaction chamber 3. The liquid inlet channel 6 is used to facilitate the addition of reaction liquid. First one-way valves 7 are arranged on both sides of the reaction chamber 3. When the first one-way valve 7 is opened, the reaction chamber 3 is connected to the cleaning pipe 2. The cleaning pipe 2 is used for the circulation of water. One end of the cleaning pipe 2 is connected to a water inlet pipe 11, and the other end of the cleaning pipe 2 is connected to a water outlet pipe 12. The water inlet pipe 11 is used for water to enter, and the water outlet pipe 12 is used for water to be discharged. The water inlet pipe 11 is provided with a peristaltic pump 13, and the peristaltic pump 13 is used to control the flow rate of water.

[0042] In further detail, the base 1 is made of transparent plastic. The transparent plastic can facilitate the operator to observe and record the experimental phenomenon.

[0043] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0044] Although the terms such as base 1, cleaning pipe 2, reaction chamber 3, opening 4, groove 5, liquid inlet channel 6, first one-way valve 7, water inlet pipe 11, water outlet pipe 12, peristaltic pump 13 are used more frequently in this article, the possibility of using other terms is not excluded. These terms are used only to more conveniently describe and explain the essence of the present invention; interpreting them as any additional limitation is contrary to the spirit of the present invention.

Claims

1. A method for detecting NR2F1 antigen, characterized in that: The steps include: Step A: preparing a glucose oxidase-modified NR2F1 second antibody solution; the preparation method of the glucose oxidase-modified NR2F1 second antibody solution in step A is: taking 10 ml of 0.2 mg / ml nano titanium dioxide solution and adding it dropwise to 8 ml of 0.1 mg / ml 3-aminopropyltrimethoxysilane methanol solution to obtain a mixed solution a; mixing the mixed solution a with 18 ml of 0.1 mg / ml nano silver sol to form a mixed solution b; adding 2 μl of 5 μg / ml NR2F1 second antibody to the mixed solution b to obtain a mixed solution c; mixing the mixed solution c with 10 ml of 0.2 mg / ml glucose oxidase to obtain a glucose oxidase-modified NR2F1 second antibody solution; Step B: preparing an electrode paper sheet for modifying a working electrode with a NR2F1 first antibody; in the step B: applying 2 μl of a 0.1 mg / ml graphene-perfluorosulfonic acid solution on the carbon electrode of the electrode paper sheet; dripping 1 μl of a 0.1 mg / ml cysteine ​​solution onto the electrode; dripping a nanosilver sol onto the electrode surface; dripping 2 μl of a 5 μg / ml NR2F1 first antibody solution onto the electrode surface, and finally drying at room temperature to form a working electrode, thereby obtaining an electrode paper sheet for modifying a working electrode with a NR2F1 first antibody; Step C: insert the electrode paper in step B into the reaction pool, drip NR2F1 antigen extract into the reaction pool, then drip the NR2F1 second antibody solution modified by glucose oxidase in step A, react for 1 to 3 minutes, drip glucose solution, react for 20 to 40 seconds, and detect the current; The reaction pool comprises a base (1), and a cleaning pipe (2) is provided through the base (1). A reaction chamber (3) is arranged in the cleaning pipe (2), an opening (4) is arranged at the top of the reaction chamber (3), a groove (5) is arranged at the top of the base (1), the opening (4) is located at the bottom of the groove (5), and the opening (4) is used for inserting one end of the electrode paper into the reaction chamber (3). The base (1) is also provided with a liquid inlet channel (6), the liquid inlet channel (6) is connected to the reaction chamber (3), and the liquid inlet channel (6) is used to facilitate the addition of reaction liquid. First one-way valves (7) are arranged on both sides of the reaction chamber (3). When When the first one-way valve (7) is opened, the reaction chamber (3) and the cleaning pipe (2) are connected. The cleaning pipe (2) is used for the circulation of water. One end of the cleaning pipe (2) is connected to a water inlet pipe (11), and the other end of the cleaning pipe (2) is connected to a water outlet pipe (12). The water inlet pipe (11) is used for the inlet of water, and the water outlet pipe (12) is used for the discharge of water. The water inlet pipe (11) is provided with a peristaltic pump (13), and the peristaltic pump (13) is used to control the flow rate of water. The base (1) is made of transparent plastic. Step D: Repeat step C by adding different concentrations of NR2F1 antigen extracts. According to the relationship between antigen concentration and current value, Prepare a standard curve; Step E: Take the sample to be tested and test it to obtain the NR2F1 antigen concentration value in the sample to be tested.

2. The method for detecting NR2F1 antigen according to claim 1, characterized in that: In step C, the electrode paper in step B is inserted into the reaction pool, 0.5 ml of NR2F1 antigen extract is added dropwise into the reaction pool, and then 1 ml of glucose oxidase-modified NR2F1 second antibody solution in step A is added dropwise. After reacting for 2 minutes, 2 ml of 0.1 mg / ml glucose solution is added dropwise. After reacting for 30 seconds, the current is detected.

3. The method for detecting NR2F1 antigen according to claim 1, characterized in that: In step C, before dripping the glucose solution, the working electrode and the reaction cell of the electrode paper need to be rinsed with deionized water.

4. The method for detecting NR2F1 antigen according to claim 1, characterized in that: In step D, the concentrations of NR2F1 antigen extracts were 5.0×10 -9 mol / L、1.0×10 -8 mol / L、5.0×10 -8 mol / L、1.0×10 -7 mol / L、5.0×10 -7 mol / L、1.0×10 -6 mol / L、1.5×10 -6 mol / L、2.0×10 -6 mol / L.

5. The method for detecting NR2F1 antigen according to claim 1, characterized in that: In step E, the electrode paper in step B is inserted into the reaction cell, the sample to be tested is added dropwise, and then 1 ml of the glucose oxidase-modified NR2F1 second antibody solution in step A is added dropwise. After reacting for 2 minutes, 2 ml of 0.1 mg / ml glucose solution is added dropwise. After reacting for 30 seconds, the current is detected and the standard curve in step D is used to calculate the NR2F1 antigen concentration value in the sample to be tested.

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