CN104133070B - Preparation method and application of a label-free immunosensor for environmental estrogen - Google Patents

Abstract

The invention discloses a preparation method of a label-free immune sensor for environmental estrogen and its application in the detection of environmental estrogen, belonging to the technical field of new nano functional materials and environmental detection. The present invention utilizes two novel nano-functional materials to modify the working electrode, utilizes the synergistic catalytic effect of the two and the adsorption effect on antibodies, thereby preparing a device for detecting environmental estrogen with high sensitivity, good specificity, rapid detection and simple preparation. Label-free electrochemical immunosensor.

Description

Preparation method and application of a label-free immunosensor for environmental estrogen

technical field

The invention relates to a preparation method and application of an environmental estrogen label-free immunosensor. Specifically, it is a label-free electrochemical immunosensor for detecting environmental estrogen constructed by a new nanocomposite material with a synergistic catalytic effect, which belongs to the technical field of new nanometer functional materials and environmental detection.

Background technique

Environmental estrogen is a kind of chemical substance that exists in the environment. It has the activity similar to that of estrogen in organisms. After entering the human body, it has the effect of simulating estrogen. It is a class of environmental toxins that seriously endanger human health. Common sources of environmental estrogen include pesticides (such as organochlorine pesticides), second-hand smoke, waste gas (such as toxic gases produced by burning plastic waste), food additives, etc.

The detection methods for environmental estrogen mainly include high performance liquid chromatography, gas chromatography, chromatography-mass spectrometry, enzyme-linked immunoassay, radioimmunoassay, etc. However, most of the detection methods have expensive instruments and complicated operations, and the inspectors must undergo strict training before they can carry out the detection. Some simple methods, such as electrochemical analysis, are simple in operation, sensitive and fast in detection, but have poor specificity. Therefore, electrochemical immunosensors that can detect environmental estrogens in the environment quickly, sensitively, and with high specificity are particularly prominent.

Electrochemical immunosensors are widely used in clinical analysis and environmental detection due to their high sensitivity, good specificity, and easy operation. They can generally be divided into two types: labeled and unlabeled. The requirements for the detection environment are stricter and the operation is relatively cumbersome. Therefore, in the detection of environmental estrogen, the label-free electrochemical immunosensor has more application prospects. To construct a label-free immunosensor for environmental estrogen, the key technology is to improve the immobilization amount of the modified electrode to the antibody and the signal response speed and size to the test bottom solution. The AuPd nanorod composite material with core-shell structure is a new type of noble metal nanocomposite material. Due to its good biocompatibility and nano-scale size, the adsorption capacity of antibodies is increased; and in the core-shell structure, Au is the core and Pd is the shell, It reduces the usage of Pd and lowers the cost; at the same time, its special rod-like shape endows Pd with more catalytic crystal faces, which can better improve the catalytic response speed and current response signal size of hydrogen peroxide used as the test bottom liquid . In addition, on the one hand, CoFe ₂ O ₄ /rGo increases the specific surface area of the electrode and enhances the conductivity of the electrode. On the other hand, it can produce a synergistic catalytic effect with AuPd, and greatly enhance the catalytic response speed and current response signal to hydrogen peroxide. Therefore, the combined use of the two can be successfully used in the construction of label-free electrochemical immunosensors.

The present invention successively uses CoFe ₂ O ₄ /rGo and AuPd-Ab to modify the electrode, and then utilizes the addition of environmental estrogen antigen to reduce the current response signal of the electrode to hydrogen peroxide, thereby realizing the construction of a label-free electrochemical immunosensor , and applied to the simple, rapid, specific and sensitive detection of environmental estrogen.

Contents of the invention

One of the purposes of the present invention is to avoid the shortcomings of existing detection methods such as complex equipment, cumbersome operation process, and poor specificity, and provide a method for preparing an environmental estrogen-free immunosensor. The prepared sensor has high sensitivity, With strong specificity, simple preparation and convenient operation, it can be used for rapid and sensitive detection of environmental estrogen;

The second object of the present invention is to apply the prepared label-free immunosensor to the detection of environmental estrogens, so as to realize rapid and sensitive detection of various environmental estrogens.

The technical scheme that the present invention adopts is as follows:

1. A preparation method of an environmental estrogen label-free immunosensor, the environmental estrogen label-free immunosensor comprising: a working electrode, a reference electrode and a counter electrode, the base electrode of the working electrode is a glassy carbon electrode, and its surface sequentially modify CoFe ₂ O ₄ /rGo, glutaraldehyde, AuPdNRs-Ab, and bovine serum albumin (BSA), the reference electrode is a saturated calomel electrode, the counter electrode is a platinum wire electrode, and the CoFe ₂ O ₄ /rGo is a cobalt ferrite/reduced graphene composite material, and the AuPtNRs-Ab is an environmental estrogen antibody incubated with a gold-palladium core-shell nanorod composite material;

It is characterized in that the preparation method comprises the following steps:

a. Preparation of CoFe ₂ O ₄ /rGo;

b. Preparation of AuPdNRs-Ab;

c. Preparation of working electrode for environmental estrogen label-free immunosensor;

Wherein, the specific steps for preparing CoFe ₂ O ₄ /rGo in step a are as follows:

Ultrasonic dispersion of 8-12 mg graphene oxide into 25-35 ml of absolute ethanol, followed by adding 0.5-1.5 ml of 0.2 mol/mL Fe(NO ₃ ) ₃ aqueous solution, 0.25-0.75 ml of 0.2 mol/L Co( NO ₃ ) ₂ aqueous solution and 2.0~3.0 ml ammonia water, stirred at 80°C for 10~14h, then transferred to a high-pressure reactor, reacted at 180°C for 4~6h; cooled to room temperature, centrifuged, and placed in a vacuum oven at 50°C Dry in medium to get CoFe ₂ O ₄ /rGo;

Step b The specific steps for preparing AuPdNRs-Ab are as follows:

Mix 20mL gold nanorod solution and 20mL 0.05~0.2 mol/L cetyltrimethylammonium bromide (CTAB) solution, stir at 30~45℃, and add 3~6 mL 0.1 mol/L L-ascorbic acid (AA) solution, 0.5-1 mL 0.03 mol/L palladium chloride (PdCl ₂ ) solution and 3-6 mL 0.1 mol/L sodium hydroxide (NaOH) solution, stop stirring, and let stand for 1 ~3h, centrifuge, add 20mL PBS buffer solution with pH7.4, and then add environmental estrogen antibody (Ab), shake at 4°C for 24h to obtain AuPdNRs-Ab;

Step c The specific steps for preparing the working electrode of the environmental estrogen label-free immunosensor are as follows:

① Pretreat the working electrode to make its surface smooth;

② Add 5~10 μL of 1~5 mg/mL CoFe ₂ O ₄ /rGo aqueous solution dropwise to the surface of the electrode obtained in ①, and let it dry naturally at room temperature;

③ Add 5-10 μL of glutaraldehyde dropwise to the surface of the electrode obtained in ②, and let it dry naturally at room temperature;

④ Add 5~8 μL of 10 μg/mL AuPdNRs-Ab solution dropwise to the surface of the electrode obtained in ③, store in a refrigerator at 4 ℃ to dry, wash with ultrapure water, dry to form a film, and store at 4 ℃;

⑤ Add 4~6 μL of 100 μg/mL BSA solution dropwise to the surface of the electrode obtained in ④ to seal the non-specific active sites on the electrode, store it in a refrigerator at 4°C and dry it, wash it with ultrapure water, and dry it in the air. Membrane, that is, the working electrode of the label-free immunosensor for environmental estrogen.

2. The environmental estrogen label-free immunosensor of the present invention is used for the detection of environmental estrogen, and the steps are as follows:

1) Add environmental estrogen antigen standard solution of known concentration to 40-60 μL PBS buffer solution with pH=7.0-8.0 to prepare antigen mixed solution, take 5-10 μL antigen mixed solution and drop-spray on the prepared On the working electrode of the environmental estrogen immunosensor, store and dry in the refrigerator at 4 ℃, wash with PBS buffer solution with pH=7.0~8.0, dry in the air, and store at 4 ℃;

2) Use the calomel electrode as the reference electrode, the platinum wire electrode as the counter electrode, and the working electrode assembled in 1) to form a three-electrode system, and connect it to the electrochemical workstation; add 10~25mL pH =7.0~8.0 PBS buffer solution and 1mL 3~6 mmol/L hydrogen peroxide (H ₂ O ₂ ) solution; detect the response of the assembled working electrode to H ₂ O ₂ by chronoamperometry; according to the obtained current response The relationship between the concentration of the environmental estrogen antigen standard solution and the working curve are drawn;

3) Replace the standard solution of the environmental estrogen antigen with the sample solution to be tested, and perform detection according to the drawing method of the working curve of the environmental estrogen antigen.

3. The preparation method and application of the environmental estrogen label-free immunosensor of the present invention, characterized in that the environmental estrogen is selected from one of the following: estradiol, estriol, diethylstilbestrol, bisphenol A or nonyl phenol.

Beneficial results of the present invention

(1) The environmental estrogen label-free immunosensor of the present invention is simple to prepare, easy to operate, realizes rapid, sensitive, and highly selective detection of various environmental estrogens, and has market development prospects.

(2) The present invention uses CoFe ₂ O ₄ /rGo and AuPdNRs-Ab to modify the electrode for the first time, and utilizes the synergistic catalytic effect of the two to significantly increase the corresponding speed and magnitude of the current, and further enhance the adsorption capacity of the antibody, thereby constructing an electrode without The labeled electrochemical immunosensor has important scientific significance and application value.

Detailed ways

Example 1 Preparation of CoFe ₂ O ₄ /rGo

8 mg of graphene oxide was ultrasonically dispersed into 25 ml of absolute ethanol, and 0.5 ml of 0.2 mol/mL Fe(NO ₃ ) ₃ aqueous solution, 0.25 ml of 0.2 mol/L Co(NO ₃ ) ₂ aqueous solution and 2.0 ml of Ammonia, stirred at 80°C for 10h, then transferred to an autoclave, reacted at 180°C for 4h; cooled to room temperature, centrifuged, and dried in a vacuum oven at 50°C to obtain CoFe ₂ O ₄ /rGo.

Example 2 Preparation of CoFe ₂ O ₄ /rGo

Ultrasonic dispersion of 10 mg of graphene oxide into 30 ml of absolute ethanol was followed by adding 1 ml of 0.2 mol/mL Fe(NO ₃ ) ₃ aqueous solution, 0.5 ml of 0.2 M Co(NO ₃ ) ₂ aqueous solution and 2.50 ml of ammonia water, Stir at 80°C for 12h, then transfer to an autoclave, react at 180°C for 5h; cool to room temperature, centrifuge and dry in a vacuum oven at 50°C to obtain CoFe ₂ O ₄ /rGo.

Example 3 Preparation of CoFe ₂ O ₄ /rGo

12 mg of graphene oxide was ultrasonically dispersed into 35 ml of absolute ethanol, and 1.5 ml of 0.2 mol/mL Fe(NO ₃ ) ₃ aqueous solution, 0.75 ml of 0.2 mol/L Co(NO ₃ ) ₂ aqueous solution and 3.0 ml of Ammonia, stirred at 80°C for 14h, then transferred to an autoclave, reacted at 180°C for 6h; cooled to room temperature, centrifuged, and dried in a vacuum oven at 50°C to obtain CoFe ₂ O ₄ /rGo.

Example 4 Preparation of AuPdNRs-Ab

Mix 20 mL of gold nanorod solution and 20 mL of 0.05 mol/L cetyltrimethylammonium bromide (CTAB) solution, stir at 30 °C, and sequentially add 3 mL of 0.1 mol/L L-ascorbic acid (AA ) solution, 0.5 mL 0.03 mol/L palladium chloride (PdCl ₂ ) solution and 3 mL 0.1 mol/L sodium hydroxide (NaOH) solution, stop stirring, let stand for 1 hour, centrifuge, add 20 mL pH7.4 PBS buffer solution, and then add environmental estrogen antibody (Ab), shake at 4°C for 24h to obtain AuPdNRs-Ab.

Example 5 Preparation of AuPdNRs-Ab

Mix 20 mL of gold nanorod solution and 20 mL of 0.1 mol/L CTAB solution, stir at 35 °C, and sequentially add 4 mL of 0.1 mol/L AA solution, 0.8 mL of 0.03 mol/L _PdCl solution and 4 mL of 0.1 mol/L NaOH solution, stop stirring, let stand for 2h, centrifuge, add 20mL PBS buffer solution with pH7.4, then add environmental estrogen antibody (Ab), shake at 4°C for 24h, and obtain AuPdNRs-Ab.

Example 6 Preparation of AuPdNRs-Ab

Mix 20 mL of gold nanorod solution and 20 mL of 0.2 mol/L CTAB solution, stir at 45 °C, and sequentially add 6 mL of 0.1 mol/L AA solution, 1 mL of 0.03 mol/L _PdCl2 solution and 6 mL of 0.1 mol/L NaOH solution, stop stirring, let stand for 3h, centrifuge, add 20mL PBS buffer solution with pH7.4, then add environmental estrogen antibody (Ab), shake at 4°C for 24h, and obtain AuPdNRs-Ab.

Example 7 Preparation method of environmental estrogen label-free immunosensor working electrode

(1) Pretreat the working electrode to make its surface smooth;

(2) Add 5 μL of 1 mg/mL CoFe ₂ O ₄ /rGo aqueous solution dropwise to the surface of the electrode obtained in (1), and let it dry naturally at room temperature;

(3) Add 5 μL of glutaraldehyde dropwise to the surface of the electrode obtained in (2), and let it dry naturally at room temperature;

(4) Add 5 μL of 10 μg/mL AuPdNRs-Ab solution dropwise to the surface of the electrode obtained in (3), store in a refrigerator at 4 °C to dry, wash with ultrapure water, dry to form a film, and store at 4 °C;

(5) Add 4 μL of 100 μg/mL BSA solution dropwise to the surface of the electrode obtained in (4) to seal the non-specific active sites on the electrode, store in a refrigerator at 4°C to dry, wash with ultrapure water, and dry in the air. After drying into a film, the working electrode of the environmental estrogen-free immunosensor can be obtained.

Example 8 Preparation Method of Environmental Estrogen Label-Free Immunosensor Working Electrode

(1) Pretreat the working electrode to make its surface smooth;

(2) Add 8 μL of 3 mg/mL CoFe ₂ O ₄ /rGo aqueous solution dropwise to the surface of the electrode obtained in (1), and let it dry naturally at room temperature;

(3) Add 8 μL of glutaraldehyde dropwise to the surface of the electrode obtained in (2), and let it dry naturally at room temperature;

(4) Add 6 μL of 10 μg/mL AuPdNRs-Ab solution dropwise to the surface of the electrode obtained in (3), store in a refrigerator at 4 °C to dry, wash with ultrapure water, dry to form a film, and store at 4 °C;

(5) Add 5 μL of 100 μg/mL BSA solution dropwise to the surface of the electrode obtained in (4) to seal the non-specific active sites on the electrode, store it in a refrigerator at 4°C and dry it, wash it with ultrapure water, and dry it in the air. After drying into a film, the working electrode of the environmental estrogen-free immunosensor can be obtained.

Example 9 Preparation method of environmental estrogen label-free immunosensor working electrode

(1) Pretreat the working electrode to make its surface smooth;

(2) Add 10 μL of 5 mg/mL CoFe ₂ O ₄ /rGo aqueous solution dropwise to the surface of the electrode obtained in (1), and let it dry naturally at room temperature;

(3) Add 10 μL of glutaraldehyde dropwise to the surface of the electrode obtained in (2), and let it dry naturally at room temperature;

(4) Add 8 μL of 10 μg/mL AuPdNRs-Ab solution dropwise to the surface of the electrode obtained in (3), store in a refrigerator at 4 °C to dry, wash with ultrapure water, dry to form a film, and store at 4 °C;

(5) Add 6 μL of 100 μg/mL BSA solution dropwise to the surface of the electrode obtained in (4) to seal the non-specific active sites on the electrode, store it in a refrigerator at 4°C and dry it, wash it with ultrapure water, and dry it in the air. After drying into a film, the working electrode of the environmental estrogen-free immunosensor can be obtained.

Example 10 The environmental estrogen label-free immunosensor prepared in Examples 1-9 is used for the detection of environmental estrogen, and the steps are as follows

(1) Add the known concentration of environmental estrogen antigen standard solution to 40 μL of PBS buffer solution with pH=7.0 to prepare the antigen mixed solution, and take 5 μL of the antigen mixed solution to drop-spray on the prepared environmental estrogen immunoassay On the working electrode of the sensor, store and dry in the refrigerator at 4 °C, wash with PBS buffer solution with pH=7.0, dry in the air, and store at 4 °C;

(2) Use the calomel electrode as the reference electrode, the platinum wire electrode as the counter electrode, and the working electrode assembled in (1) to form a three-electrode system, and connect it to the electrochemical workstation; add 10mL of pH =7.0 PBS buffer solution and 1mL 3 mmol/L H ₂ O ₂ solution; detect the response of the assembled working electrode to H ₂ O ₂ by chronoamperometry; The relationship between, draw the working curve;

(3) Replace the standard solution of the environmental estrogen antigen with the sample solution to be tested, and perform detection according to the drawing method of the working curve of the environmental estrogen antigen.

Example 11 The environmental estrogen label-free immunosensor prepared in Examples 1-9 is used for the detection of environmental estrogen, and the steps are as follows

(1) Add the known concentration of environmental estrogen antigen standard solution to 50 μL of PBS buffer solution with pH=7.4 to prepare the antigen mixed solution, take 7 μL of the antigen mixed solution and drop-spray it on the prepared environmental estrogen immunoassay On the working electrode of the sensor, store and dry in the refrigerator at 4 °C, wash with PBS buffer solution with pH=7.4, dry in the air, and store at 4 °C;

(2) Use the calomel electrode as the reference electrode, the platinum wire electrode as the counter electrode, and the working electrode assembled in (1) to form a three-electrode system, and connect it to the electrochemical workstation; add 20mL of pH =7.4 PBS buffer solution and 1mL 4 mmol/L H ₂ O ₂ solution; detect the response of the assembled working electrode to H ₂ O ₂ by chronoamperometry; The relationship between, draw the working curve;

(3) Replace the standard solution of the environmental estrogen antigen with the sample solution to be tested, and perform detection according to the drawing method of the working curve of the environmental estrogen antigen.

Example 12 The environmental estrogen label-free immunosensor prepared in Examples 1-9 is used for the detection of environmental estrogen, and the steps are as follows

(1) Add the known concentration of environmental estrogen antigen standard solution to 60 μL of PBS buffer solution with pH=8.0 to prepare the antigen mixed solution, take 10 μL of the antigen mixed solution and drop-spray it on the prepared environmental estrogen immunoassay On the working electrode of the sensor, store and dry in the refrigerator at 4 °C, wash with PBS buffer solution with pH=8.0, dry in the air, and store at 4 °C;

(2) The calomel electrode used as the reference electrode, the platinum wire electrode used as the counter electrode, and the working electrode assembled in (1) form a three-electrode system, which is connected to the electrochemical workstation; 25mL pH =8.0 PBS buffer solution and 1mL 6 mmol/L H ₂ O ₂ solution; detect the response of the assembled working electrode to H ₂ O ₂ by chronoamperometry; The relationship between, draw the working curve;

(3) Replace the standard solution of the environmental estrogen antigen with the sample solution to be tested, and perform detection according to the drawing method of the working curve of the environmental estrogen antigen.

Embodiment 13 environmental estrogen: detection of estradiol, estriol, diethylstilbestrol, bisphenol A or nonylphenol, comprising the following steps

(1) Select an environmental estrogen, and construct an environmental estrogen label-free immunosensor according to the steps described in Examples 1-9;

(2) Perform detection according to the steps described in Examples 10-12, and see Table 1 for the technical indicators of environmental estrogen detection.

Table 1 Detection technical indicators of environmental estrogen

The detection of environmental estrogen in embodiment 14 milk

Accurately pipette the milk sample, add a certain mass concentration of the environmental estrogen antigen standard solution, take the milk without the environmental estrogen antigen as the blank, carry out the standard addition recovery experiment, and perform detection according to the steps in Example 13 to determine the environmental concentration in the milk sample. The recovery rate of estrogen, the test results are shown in Table 2.

Table 2 Detection results of various environmental estrogens in milk

It can be seen from the detection results in Table 2 that the relative standard deviation (RSD) of the results is less than 3.0%, and the average recovery rate is 96.0-102%, which shows that the present invention can be used for the detection of various environmental estrogens in milk, and the method has high sensitivity and strong specificity , the result is accurate and reliable.

The detection of environmental estrogen in embodiment 15 water samples

Accurately pipette a certain water sample, add a certain mass concentration of environmental estrogen antigen standard solution, take the water sample without environmental estrogen antigen as a blank, carry out the standard addition recovery experiment, and perform detection according to the steps in Example 13, and measure the water sample The recovery rate of estrogen in the environment, the test results are shown in Table 3.

Table 3 Detection results of various environmental estrogens in water samples

As can be seen from the test results in Table 3, the relative standard deviation (RSD) of the results is less than 3.3%, and the average recovery rate is 97.2 to 102%, indicating that the present invention can be used for the detection of various environmental estrogens in water samples, and the method has high sensitivity and specificity Strong, accurate and reliable results.

Claims (2)

1. A preparation method of an environmental estrogen label-free immunosensor, the environmental estrogen label-free immunosensor comprising: a working electrode, a reference electrode and a counter electrode, the base electrode of the working electrode is a glassy carbon electrode, and its surface sequentially modify CoFe ₂ O ₄ /rGo, glutaraldehyde, AuPdNRs-Ab, and bovine serum albumin BSA, the reference electrode is a saturated calomel electrode, the counter electrode is a platinum wire electrode, and the CoFe ₂ O ₄ /rGo It is a cobalt ferrite/reduced graphene composite material, and the AuPtNRs-Ab is an environmental estrogen antibody hatched by a gold-palladium core-shell nanorod composite material; It is characterized in that the preparation method comprises the following steps: a. Preparation of CoFe ₂ O ₄ /rGo; b. Preparation of AuPdNRs-Ab; c. Preparation of working electrode for environmental estrogen label-free immunosensor; Wherein, the specific steps for preparing CoFe ₂ O ₄ /rGo in step a are as follows: Ultrasonic dispersion of 8-12 mg graphene oxide into 25-35 ml of absolute ethanol, followed by adding 0.5-1.5 ml of 0.2 mol/mL Fe(NO ₃ ) ₃ aqueous solution, 0.25-0.75 ml of 0.2 mol/L Co( NO ₃ ) ₂ aqueous solution and 2.0~3.0 ml ammonia water, stirred at 80°C for 10~14h, then transferred to a high-pressure reactor, reacted at 180°C for 4~6h; cooled to room temperature, centrifuged, and placed in a vacuum oven at 50°C Dry in medium to get CoFe ₂ O ₄ /rGo; Step b The specific steps for preparing AuPdNRs-Ab are as follows: Mix 20mL gold nanorod solution and 20mL 0.05~0.2 mol/L cetyltrimethylammonium bromide solution, stir at 30~45℃, and add 3~6 mL 0.1 mol/L L- Ascorbic acid solution, 0.5-1 mL 0.03 mol/L palladium chloride solution and 3-6 mL 0.1 mol/L sodium hydroxide solution, stop stirring, let stand for 1-3 hours, centrifuge, add 20 mL pH7.4 PBS Buffer solution, then add environmental estrogen antibody Ab, and shake at 4°C for 24 hours to obtain AuPdNRs-Ab; Step c The specific steps for preparing the working electrode of the environmental estrogen label-free immunosensor are as follows: ① Pretreat the working electrode to make its surface smooth; ② Add 5~10 μL of 1~5 mg/mL CoFe ₂ O ₄ /rGo aqueous solution dropwise to the surface of the electrode obtained in ①, and let it dry naturally at room temperature; ③ Add 5-10 μL of glutaraldehyde dropwise to the surface of the electrode obtained in ②, and let it dry naturally at room temperature; ④ Add 5~8 μL of 10 μg/mL AuPdNRs-Ab solution dropwise to the surface of the electrode obtained in ③, store in a refrigerator at 4 ℃ to dry, wash with ultrapure water, dry to form a film, and store at 4 ℃; ⑤ Add 4~6 μL of 100 μg/mL BSA solution dropwise to the surface of the electrode obtained in ④ to seal the non-specific active sites on the electrode, store it in a refrigerator at 4°C and dry it, wash it with ultrapure water, and dry it in the air. Membrane, that is, the working electrode of the label-free immunosensor for environmental estrogen. 2. The label-free immunosensor for environmental estrogen as claimed in claim 1 is used for the detection of environmental estrogen, and the steps are as follows: 1) Add environmental estrogen antigen standard solution of known concentration to 40-60 μL PBS buffer solution with pH=7.0-8.0 to prepare antigen mixed solution, take 5-10 μL antigen mixed solution and drop-spray on the prepared On the working electrode of the environmental estrogen immunosensor, store and dry in the refrigerator at 4 ℃, wash with PBS buffer solution with pH=7.0~8.0, dry in the air, and store at 4 ℃; 2) Use the calomel electrode as the reference electrode, the platinum wire electrode as the counter electrode, and the working electrode assembled in 1) to form a three-electrode system, and connect it to the electrochemical workstation; add 10~25mL pH =7.0~8.0 PBS buffer solution and 1mL 3~6 mmol/L hydrogen peroxide H ₂ O ₂ solution; detect the response of the assembled working electrode to H ₂ O ₂ by chronoamperometry; according to the obtained current response and the environment The relationship between the concentration of estrogen antigen standard solution, draw the working curve; 3) Replace the standard solution of the environmental estrogen antigen with the sample solution to be tested, and perform detection according to the drawing method of the working curve of the environmental estrogen antigen. 3. The preparation method of environmental estrogen label-free immunosensor as claimed in claim 1, characterized in that the environmental estrogen is selected from one of the following: estradiol, estriol, diethylstilbestrol, bisphenol A or nonyl phenol.