Preparation method and application of ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB

Abstract

The present invention belongs to the field of new functional materials and biosensor technology, and relates to the preparation of an electrochemical sensor based on ferrous selenide-gold nanoparticles (FeSe2-Au NPs), which is used for sensitive detection of biological serum cytokeratin 19 fragment antigen 21-1 (CYFRA21-1). A composite material of FeSe2-Au NPs as a substrate and a composite material of toluidine blue loaded on polyacrylic acid functionalized ZIF-67 (PAA-ZIF-TB) as a marker are prepared respectively, and a sandwich-type immunosensor is constructed based on the composite material, and a ratio type (differential pulse voltammetry) is used for detection. The electrochemical immunosensor constructed according to this method is used to determine the concentration of CYFRA21-1 in actual serum samples, and exhibits excellent stability and sensitivity, providing a new detection method for detecting CYFRA21-1.

Description

Technical Field

[0001] The invention relates to the preparation and application of an electrochemical immunosensor, in particular to a sandwich-type immunosensor using FeSe2-Au NPs as a sensing platform and PAA-ZIF-TB as a marker. The invention belongs to the field of new functional materials and biosensor technology. Background Art

[0002] Lung cancer is one of the most threatening malignant tumors to human health and life, accounting for 13% of all cancer cases. It is the leading cause of cancer death worldwide and one of the major global public health issues. Lung cancer is relatively latent in its initial onset, with no characteristic clinical symptoms in the early stages. 80% of lung cancer patients have already developed to the middle or late stages when they are diagnosed in the hospital, missing the best time for treatment. Therefore, early detection and diagnosis of lung cancer are particularly important in the entire treatment. Serum cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) is a biomarker for detecting tumors and is currently widely used in the diagnosis of cancer. We need to develop new detection methods to complete the detection of CYFRA21-1.

[0003] Electrochemical immunosensor is a more advanced analytical detection technology based on immunosensor, and it is the earliest and more mature branch of immunosensor research. Electrochemical immunosensor combines a highly sensitive electrochemical detection system with a highly selective immune recognition system and is widely used in the detection of biomarkers. Electrochemical immunosensor has the advantages of high selectivity, high sensitivity and low detection limit, and is widely used in immunoanalysis.

[0004] In this work, FeSe2 was used as an electrochemical signal probe in biosensors. It has excellent EC performance and strong signal output, and was successfully applied to electrochemical biosensors. However, the disadvantage is that the conductivity of FeSe2 is low. Loading Au NPs on the surface of FeSe2 can improve the conductivity of the material, and there are a large number of active sites on the surface of Au NPs that can fix antibodies. Therefore, we prepared FeSe2-Au NPs as the matrix of biosensors. Electroactive toluidine blue (TB) is a phenothiazine cationic dye that can undergo redox reactions. It is a good choice to use it as a redox probe for electrochemical biosensors. ZIF-67 is composed of Co cations and imidazole ligands. As a member of the ZIF family, it has a large specific surface area, controllable porosity and more targeting sites and has attracted widespread attention. The application prospects in the field of adsorption are very promising. It has been reported that ZIF-67 has good adsorption properties for cations. ZIF-67 functionalized with polyacrylic acid (PAA) is electronegative and binds to TB through electrostatic adsorption, increasing the adsorption amount of TB and improving the sensitivity of the immunosensor. The electrochemical immunosensor constructed according to this method was used to determine the CYFRA21-1 concentration in actual serum samples, showing excellent stability and selectivity, providing a new method for the detection of CYFRA21-1. Summary of the invention

[0005] One of the purposes of the present invention is to prepare a sandwich immunosensor using FeSe2-Au NPs as a sensing platform and PAA-ZIF-TB as a marker.

[0006] The second purpose of the present invention is to use the sensor for highly sensitive and specific detection of CYFRA21-1.

[0007] The technical solution of the present invention is as follows:

[0008] 1. A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB, comprising the following steps:

[0009] (1) Polish the glassy carbon electrode with polishing powder, then wash it with deionized water, place the electrode in a 5 mmol / L potassium ferrocyanide solution, and scan it at a potential of -0.6 to 0.6 V until the peak potential difference is less than 110 mV;

[0010] (2) 6 μL of 2 ~ 4 mg / mL FeSe2-Au NPs solution was dropped onto the electrode and dried at room temperature;

[0011] (3) 6 μL of 1 ~ 3 μg / mL serum cytokeratin 19 fragmentantigen 21-1 (CYFRA21-1) primary antibody was added to the electrode, dried at room temperature, washed with PBS to remove excess antibody, and dried at room temperature;

[0012] (4) Add 3 μL of 1-2% BSA solution onto the electrode to block nonspecific binding sites. After drying, wash away excess BSA with PBS and dry at room temperature.

[0013] (5) 6 μL of a series of CYFRA21-1 antigen standard solutions with different concentrations ranging from 0.1 pg / mL to 100 ng / mL was dripped onto the electrode, dried at room temperature, washed with PBS to remove excess antigen, and dried at room temperature;

[0014] (6) 6 μL of 1 ~ 5 mg / mL PAA-ZIF-TB-Ab2 was added to the electrode, dried at room temperature, washed with PBS to remove excess secondary antibody, and dried at room temperature.

[0015] 2. A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB. The preparation steps of FeSe2-Au NPs are as follows:

[0016] (1) Synthesis of Au NPs

[0017] Place 100-300 mL of 0.01 wt% HAuCl4 solution in an oil bath until it is boiling, then add 1.5-3.5 mL of trisodium citrate solution. When the solution turns reddish brown, stop heating and cool to room temperature to obtain the desired Au NPs.

[0018] (2) Synthesis of FeSe2

[0019] Fe2O3 was prepared by a hydrothermal method. 97.8 ~ 99.8 mg K3[Fe(CN)6] was dissolved in 10 ~ 30 mL deionized water and stirred for 10 minutes. The solution was transferred to a 50 mL polytetrafluoroethylene autoclave and reacted at 140 °C for 48 h. The solution was washed with deionized water several times and dried in a 60 °C oven overnight. Next, 1 ~ 2 mol Se powder and 10 ~ 30 mg Fe2O3 were added to 10 ~ 30 mL oleylamine solution and stirred for 10 minutes. The solution was heated to 200 °C and kept under magnetic stirring for one hour. The solution was washed with deionized water and ethanol several times and dried in a oven overnight.

[0020] (3) Synthesis of FeSe2-Au NPs

[0021] Subsequently, FeSe2 and 10 ~ 30mL Au NPs solution were mixed, ultrasonicated for 1 h, shaken for 24 h, washed with deionized water and ethanol for multiple times, and finally dried in a drying oven to obtain FeSe2-Au NPs powder.

[0022] 3. A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and ZIF-PAA-TB. The preparation steps of ZIF-PAA-TB are as follows:

[0023] (1) Synthesis of PAA-ZIF-67

[0024] PAA-ZIF-67 was synthesized by room temperature stirring scheme. 0.35 ~ 0.55 g of cobalt nitrate hexahydrate solution was added to 2 ~ 4 mL of deionized water by magnetic stirring to form solution A, 4.5 g ~ 6.5 g of 2-methylimidazole was added to 10 ~ 20 mL of deionized water to form solution B, 40 ~ 60 μL of PAA was added to solution A and stirred for 10 minutes. The two solutions were mixed and stirred for 6 h, washed with water and methanol several times, and dried in a vacuum oven at 60 °C for 12 h to obtain a purple powder;

[0025] (2) Synthesis of PAA-ZIF-TB

[0026] 30-50 mg PAA-ZIF-67 was dissolved in 30-40 mL H2O and sonicated for 30 min, then 3-5 mL 1 mg / mL TB solution was added, shaken at room temperature for 24 h, collected by centrifugation, washed several times with ultrapure water, and finally dried overnight to obtain PAA-ZIF-TB;

[0027] (3) Synthesis of PAA-ZIF-TB-Ab2

[0028] Weigh 5 mg of PAA-ZIF-TB, add 1 mL of PBS (pH = 7.38) to fully dissolve it, then add 200 μL of 1 μg / mL CYFRA21-1-Ab2, oscillate at 4 ℃ for 24 h, centrifuge at 4 ℃, and add 1 mL of PBS solution to the obtained precipitate to obtain PAA-ZIF-TB-Ab2.

[0029] 4. Application of a preparation method of a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and ZIF-PAA-TB. The detection steps of CYFRA21-1 are as follows:

[0030] (1) The three-electrode system of the electrochemical workstation was used for testing. All electrochemical measurements adopted the traditional three-electrode system: a saturated calomel electrode was used as the reference electrode, a platinum electrode was used as the counter electrode, and the prepared electrochemical immunosensor was used as the working electrode. The electrochemical measurements were carried out in PBS buffer with a pH of 7.38 by the DPV method. The parameters were set as follows: range and direction: -0.6 ~ 0.6 V, potential increment: 0.004 V, frequency: 15 Hz, amplitude: 0.05 V.

[0031] (2) In 10 mL of PBS buffer solution with a pH of 6.0 to 9.2, the electrochemical signals generated by different concentrations of CYFRA21-1 standard solutions were detected by electrochemical method, and a working curve was drawn;

[0032] (3) The CYFRA21-1 sample solution to be tested is used instead of the CYFRA21-1 standard solution for determination.

[0033] Beneficial results of the present invention:

[0034] The present invention adopts FeSe2-Au NPs as a substrate material, which has excellent electrochemical activity and low charge transfer resistance, and can bind antibodies to a greater extent, has a high and stable electrochemical signal, and improves the sensitivity of detecting CYFRA21-1; PAA-ZIF-TB is used as a marker and incubated with a secondary antibody, showing excellent electrochemical performance; an ultrasensitive electrochemical immunosensor constructed using FeSe2-Au NPs and PAA-ZIF-TB composite materials can be applied to clinical detection of CYFRA21-1, and has the advantages of simple operation, rapid detection, wide signal linear range (0.1 pg / mL ~ 100 ng / mL) and low detection limit (0.05 pg / mL). DETAILED DESCRIPTION

[0035] Example 1 A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB, the preparation steps of FeSe2-Au NPs are as follows:

[0036] (1) Synthesis of Au NPs

[0037] Place 100 mL of 0.01 wt% HAuCl4 solution in an oil bath until it is boiling, then add 1.5 mL of trisodium citrate solution. When the solution turns reddish brown, stop heating and cool to room temperature to obtain the desired Au NPs.

[0038] (2) Synthesis of FeSe2

[0039] Fe2O3 was prepared by hydrothermal method. 97.8 mg K3[Fe(CN)6] was dissolved in 10 mL deionized water and stirred for 10 minutes. The solution was transferred to a 50 mL polytetrafluoroethylene autoclave and reacted at 140 °C for 48 h. The solution was washed with deionized water several times and dried in a 60 °C oven overnight. Next, 1 mol Se powder and 10 mg Fe2O3 were added to 10 mL oleylamine solution and stirred for 10 minutes. The solution was heated to 200 °C and kept under magnetic stirring for one hour. The solution was washed with deionized water and ethanol several times and dried in a oven overnight.

[0040] (3) Synthesis of FeSe2-Au NPs

[0041] Subsequently, FeSe2 and 10 mL of Au NPs solution were mixed, ultrasonicated for 1 h, shaken for 24 h, washed with deionized water and ethanol for multiple times, and finally dried in a drying oven to obtain FeSe2-Au NPs powder.

[0042] Example 2 A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB, the preparation steps of FeSe2-Au NPs are as follows:

[0043] (1) Synthesis of Au NPs

[0044] 200 mL of 0.01 wt% HAuCl4 solution was brought to a boil in an oil bath, and then 2.5 mL of trisodium citrate solution was added. When the solution turned red-brown, heating was stopped and cooled to room temperature to obtain the desired Au NPs.

[0045] (2) Synthesis of FeSe2

[0046] Fe2O3 was prepared by hydrothermal method. 98.8 mg K3[Fe(CN)6] was dissolved in 20 mL deionized water and stirred for 10 minutes. The solution was transferred to a 50 mL polytetrafluoroethylene autoclave and reacted at 140 °C for 48 h. The solution was washed with deionized water several times and dried in a 60 °C oven overnight. Next, 1.5 mol Se powder and 20 mg Fe2O3 were added to 20 mL oleylamine solution and stirred for 10 minutes. The solution was heated to 200 °C and kept under magnetic stirring for one hour. The solution was washed with deionized water and ethanol several times and dried in a dry box overnight.

[0047] (3) Synthesis of FeSe2-Au NPs

[0048] Subsequently, FeSe2 and 20 mL of Au NPs solution were mixed, ultrasonicated for 1 h, shaken for 24 h, washed with deionized water and ethanol for multiple times, and finally dried in a drying oven to obtain FeSe2-Au NPs powder.

[0049] Example 3 A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB, the preparation steps of FeSe2-Au NPs are as follows:

[0050] (1) Synthesis of Au NPs

[0051] 300 mL of 0.01 wt% HAuCl4 solution was brought to a boil in an oil bath, and then 3.5 mL of trisodium citrate solution was added. When the solution turned red-brown, heating was stopped and cooled to room temperature to obtain the desired Au NPs.

[0052] (2) Synthesis of FeSe2

[0053] Fe2O3 was prepared by hydrothermal method. 99.8 mg K3[Fe(CN)6] was dissolved in 30 mL deionized water and stirred for 10 minutes. The solution was transferred to a 50 mL polytetrafluoroethylene autoclave and reacted at 140 °C for 48 h. The solution was washed with deionized water several times and dried in a 60 °C oven overnight. Next, 2 mol Se powder and 30 mg Fe2O3 were added to 30 mL oleylamine solution and stirred for 10 minutes. The solution was heated to 200 °C and kept under magnetic stirring for one hour. The solution was washed with deionized water and ethanol several times and dried in a dry box overnight.

[0054] (3) Synthesis of FeSe2-Au NPs

[0055] Subsequently, FeSe2 and 30 mL of Au NPs solution were mixed, ultrasonicated for 1 h, shaken for 24 h, washed with deionized water and ethanol for multiple times, and finally dried in a drying oven to obtain FeSe2-Au NPs powder.

[0056] Example 4 A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and ZIF-PAA-TB, the preparation steps of ZIF-PAA-TB are as follows:

[0057] (1) Synthesis of PAA-ZIF-67

[0058] PAA-ZIF-67 was synthesized by room temperature stirring scheme. 0.35 g of cobalt nitrate hexahydrate solution was added to 2 mL of deionized water by magnetic stirring to form solution A, 4.5 g of 2-methylimidazole was added to 10 mL of deionized water to form solution B, 40 μL of PAA was added to solution A and stirred for 10 min, the two solutions were mixed and stirred for 6 h, washed with water and methanol several times, and dried in a vacuum oven at 60 °C for 12 h to obtain a purple powder;

[0059] (2) Synthesis of PAA-ZIF-TB

[0060] 30 mg of PAA-ZIF-67 was dissolved in 30 mL of H2O and sonicated for 30 min, then 3 mL of 1 mg / mL TB solution was added, shaken at room temperature for 24 h, collected by centrifugation, washed several times with ultrapure water, and finally dried overnight to obtain PAA-ZIF-TB;

[0061] (3) Synthesis of PAA-ZIF-TB-Ab2

[0062] Weigh 5 mg of PAA-ZIF-TB, add 1 mL of PBS (pH=7.38) to fully dissolve it, then add 200 μL of CYFRA21-1-Ab2 (1 μg / mL), oscillate at 4 °C for 24 h, centrifuge at 4 °C, and add 1 mL of PBS solution to the precipitate to obtain PAA-ZIF-TB-Ab2.

[0063] Example 5 A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and ZIF-PAA-TB, the preparation steps of ZIF-PAA-TB are as follows:

[0064] (1) Synthesis of PAA-ZIF-67

[0065] PAA-ZIF-67 was synthesized by room temperature stirring scheme. 0.45 g of cobalt nitrate hexahydrate solution was added to 3 mL of deionized water by magnetic stirring to form solution A, 5.5 g of 2-methylimidazole was added to 15 mL of deionized water to form solution B, 50 μL of PAA was added to solution A and stirred for 10 min, the two solutions were mixed and stirred for 6 h, washed with water and methanol several times, and dried in a vacuum oven at 60 °C for 12 h to obtain a purple powder;

[0066] (2) Synthesis of PAA-ZIF-TB

[0067] 40 mg of PAA-ZIF-67 was dissolved in 35 mL of H2O and sonicated for 30 min, then 4 mL of 1 mg / mL TB solution was added, shaken at room temperature for 24 h, collected by centrifugation, washed several times with ultrapure water, and finally dried overnight to obtain PAA-ZIF-TB.

[0068] (3) Synthesis of PAA-ZIF-TB-Ab2

[0069] Weigh 5 mg of PAA-ZIF-TB, add 1 mL of PBS (pH=7.38) to fully dissolve it, then add 200 μL of CYFRA21-1-Ab2 (1 μg / mL), oscillate at 4 °C for 24 h, centrifuge at 4 °C, and add 1 mL of PBS solution to the precipitate to obtain PAA-ZIF-TB-Ab2.

[0070] Example 6 A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and ZIF-PAA-TB, the preparation steps of ZIF-PAA-TB are as follows:

[0071] (1) Synthesis of PAA-ZIF-67

[0072] PAA-ZIF-67 was synthesized by room temperature stirring scheme. 0.55 g of cobalt nitrate hexahydrate solution was added to 4 mL of deionized water by magnetic stirring to form solution A, 6.5 g of 2-methylimidazole was added to 20 mL of deionized water to form solution B, 60 μL of PAA was added to solution A and stirred for 10 min, the two solutions were mixed and stirred for 6 h, washed with water and methanol several times, and dried in a vacuum oven at 60 °C for 12 h to obtain a purple powder;

[0073] (2) Synthesis of PAA-ZIF-TB

[0074] 50 mg of PAA-ZIF-67 was dissolved in 40 mL of H2O and sonicated for 30 min, then 5 mL of 1 mg / mL TB solution was added, shaken at room temperature for 24 h, collected by centrifugation, washed several times with ultrapure water, and finally dried overnight to obtain PAA-ZIF-TB;

[0075] (3) Synthesis of PAA-ZIF-TB-Ab2

[0076] Weigh 5 mg of PAA-ZIF-TB, add 1 mL of PBS (pH=7.38) to fully dissolve it, then add 200 μL of CYFRA21-1-Ab2 (1 μg / mL), oscillate at 4 °C for 24 h, centrifuge at 4 °C, and add 1 mL of PBS solution to the precipitate to obtain PAA-ZIF-TB-Ab2.

[0077] Example 7 A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB, the steps are as follows:

[0078] (1) Polish the glassy carbon electrode with polishing powder, then wash it with deionized water, place the electrode in a 5 mmol / L potassium ferrocyanide solution, and scan it at a potential of -0.6 to 0.6 V until the peak potential difference is less than 110 mV;

[0079] (2) 6 μL of 2 mg / mL FeSe2-Au NPs solution was dropped onto the electrode and dried at room temperature;

[0080] (3) 6 μL, 1 μg / mL serum cytokeratin 19 fragmentantigen 21-1 (CYFRA21-1) primary antibody was added to the electrode, dried at room temperature, washed with PBS to remove excess antibody, and dried at room temperature;

[0081] (4) Add 3 μL of 1% BSA solution onto the electrode to block nonspecific binding sites. After drying, wash away excess BSA with PBS and dry at room temperature.

[0082] (5) 6 μL of a series of CYFRA21-1 antigen standard solutions with different concentrations ranging from 0.1 pg / mL to 100 ng / mL was dripped onto the electrode, dried at room temperature, washed with PBS to remove excess antigen, and dried at room temperature;

[0083] (6) 6 μL of 1 mg / mL PAA-ZIF-TB-Ab2 was added to the electrode, dried at room temperature, washed with PBS to remove excess secondary antibody, and dried at room temperature.

[0084] Example 8 A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB, the steps are as follows:

[0085] (1) Polish the glassy carbon electrode with polishing powder, then wash it with deionized water, place the electrode in a 5 mmol / L potassium ferrocyanide solution, and scan it at a potential of -0.6 to 0.6 V until the peak potential difference is less than 110 mV;

[0086] (2) 6 μL of 3 mg / mL FeSe2-Au NPs solution was dropped onto the electrode and dried at room temperature;

[0087] (3) 6 μL of 2 μg / mL serum cytokeratin 19 fragmentantigen 21-1 (CYFRA21-1) primary antibody was added to the electrode, dried at room temperature, washed with PBS to remove excess antibody, and dried at room temperature;

[0088] (4) Add 3 μL of 1.5% BSA solution onto the electrode to block nonspecific binding sites. After drying, wash away excess BSA with PBS and dry at room temperature.

[0089] (5) 6 μL of a series of CYFRA21-1 antigen standard solutions with different concentrations ranging from 0.1 pg / mL to 100 ng / mL was dripped onto the electrode, dried at room temperature, washed with PBS to remove excess antigen, and dried at room temperature;

[0090] (6) 6 μL of 3 mg / mL PAA-ZIF-TB-Ab2 was added to the electrode, dried at room temperature, washed with PBS to remove excess secondary antibody, and dried at room temperature.

[0091] Example 9 A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB, the steps are as follows:

[0092] (1) Polish the glassy carbon electrode with polishing powder, then wash it with deionized water, place the electrode in a 5 mmol / L potassium ferrocyanide solution, and scan it at a potential of -0.6 to 0.6 V until the peak potential difference is less than 110 mV;

[0093] (2) 6 μL, 4 mg / mL FeSe2-Au NPs solution was dropped onto the electrode and dried at room temperature;

[0094] (3) 6 μL, 3 μg / mL serum cytokeratin 19 fragmentantigen 21-1 (CYFRA21-1) primary antibody was added to the electrode, dried at room temperature, washed with PBS to remove excess antibody, and dried at room temperature;

[0095] (4) Add 3 μL of 2% BSA solution onto the electrode to block nonspecific binding sites. After drying, wash away excess BSA with PBS and dry at room temperature.

[0096] (5) 6 μL of a series of CYFRA21-1 antigen standard solutions with different concentrations ranging from 0.1 pg / mL to 100 ng / mL was dripped onto the electrode, dried at room temperature, washed with PBS to remove excess antigen, and dried at room temperature;

[0097] (6) 6 μL, 5 mg / mL PAA-ZIF-TB-Ab2 was added to the electrode, dried at room temperature, washed with PBS to remove excess secondary antibody, and dried at room temperature.

[0098] Example 10 Application of a preparation method of a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and ZIF-PAA-TB, the detection steps of CYFRA21-1 are as follows:

[0099] (1) The three-electrode system of the electrochemical workstation was used for testing. All electrochemical measurements adopted the traditional three-electrode system: a saturated calomel electrode was used as the reference electrode, a platinum electrode was used as the counter electrode, and the prepared electrochemical immunosensor was used as the working electrode. The electrochemical measurements were performed in PBS buffer with a pH of 7.38 by the DPV method (range and direction: -0.6 ~ 0.6 V, potential increment: 0.004 V, frequency: 15 Hz, amplitude: 0.05 V).

[0100] (2) In 10 mL of PBS buffer solution with a pH of 6.0 to 9.2, the electrochemical signals generated by different concentrations of CYFRA21-1 standard solutions were detected by electrochemical method, and a working curve was drawn;

[0101] (3) The CYFRA21-1 sample solution to be tested is used instead of the CYFRA21-1 standard solution for determination.

Claims

1. A method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB, characterized in that Here are the steps: (1) Polish the glassy carbon electrode with polishing powder, then wash it with deionized water, place the electrode in a 5 mmol / L potassium ferrocyanide solution, and scan it at a potential of -0.6 to 0.6 V until the peak potential difference is less than 110 mV; (2) 6 μL of 2 ~ 4 mg / mL FeSe2-Au NPs solution was dropped onto the electrode and dried at room temperature; (3) 6 μL of 1 ~ 3 μg / mL serum cytokeratin 19 fragmentantigen 21-1 (CYFRA21-1) primary antibody was added to the electrode, dried at room temperature, washed with PBS to remove excess antibody, and dried at room temperature; (4) Add 3 μL of 1-2% BSA solution onto the electrode to block nonspecific binding sites. After drying, wash away excess BSA with PBS and dry at room temperature. (5) 6 μL of a series of CYFRA21-1 antigen standard solutions with different concentrations ranging from 0.1 pg / mL to 100 ng / mL was dripped onto the electrode, dried at room temperature, washed with PBS to remove excess antigen, and dried at room temperature; (6) 6 μL of 1 ~ 5 mg / mL PAA-ZIF-TB-Ab2 was added to the electrode, dried at room temperature, washed with PBS to remove excess secondary antibody, and dried at room temperature.

2. The method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB according to claim 1, characterized in that The preparation steps of FeSe2-Au NPs are as follows: (1) Synthesis of Au NPs Place 100-300 mL of 0.01 wt% HAuCl4 solution in an oil bath until it is boiling, then add 1.5-3.5 mL of trisodium citrate solution. When the solution turns reddish brown, stop heating and cool to room temperature to obtain the desired Au NPs. (2) Synthesis of FeSe2 Fe2O3 was prepared by a hydrothermal method. 97.8 ~ 99.8 mg K3[Fe(CN)6] was dissolved in 10 ~ 30 mL deionized water and stirred for 10 minutes. The solution was transferred to a 50 mL polytetrafluoroethylene autoclave and reacted at 140 °C for 48 h. The solution was washed with deionized water several times and dried in a 60 °C oven overnight. Next, 1 ~ 2 mol Se powder and 10 ~ 30 mg Fe2O3 were added to 10 ~ 30 mL oleylamine solution and stirred for 10 minutes. The solution was heated to 200 °C and kept under magnetic stirring for one hour. The solution was washed with deionized water and ethanol several times and dried in a oven overnight. (3) Synthesis of FeSe2-Au NPs Subsequently, FeSe2 and 10 ~ 30mL Au NPs solution were mixed, ultrasonicated for 1 h, shaken for 24 h, washed with deionized water and ethanol for multiple times, and finally dried in a drying oven to obtain FeSe2-Au NPs powder.

3. The method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB according to claim 1, characterized in that The preparation steps of PAA-ZIF-TB are as follows: (1) Synthesis of PAA-ZIF-67 PAA-ZIF-67 was synthesized by room temperature stirring scheme. 0.35 ~ 0.55 g of cobalt nitrate hexahydrate solution was added to 2 ~ 4 mL of deionized water by magnetic stirring to form solution A, 4.5 g ~ 6.5 g of 2-methylimidazole was added to 10 ~ 20 mL of deionized water to form solution B, 40 ~ 60 μL of PAA was added to solution A and stirred for 10 min. The two solutions were mixed and stirred for 6 h, washed with water and methanol several times, and dried in a vacuum oven at 60 °C for 12 h to obtain a purple powder. (2) Synthesis of PAA-ZIF-TB 30-50 mg PAA-ZIF-67 was dissolved in 30-40 mL H2O and sonicated for 30 min, then 3-5 mL 1 mg / mL TB solution was added, shaken at room temperature for 24 h, collected by centrifugation, washed several times with ultrapure water, and finally dried overnight to obtain PAA-ZIF-TB; (3) Synthesis of PAA-ZIF-TB-Ab2 Weigh 5 mg of PAA-ZIF-TB, add 1 mL of PBS (pH = 7.38) to fully dissolve it, then add 200 μL of 1 μg / mL CYFRA21-1-Ab2, oscillate at 4 ℃ for 24 h, centrifuge at 4 ℃, and add 1 mL of PBS solution to the obtained precipitate to obtain PAA-ZIF-TB-Ab2.

4. The use of the method for preparing a ratiometric electrochemical immunosensor based on FeSe2-Au NPs and PAA-ZIF-TB as claimed in claim 1, characterized in that The detection steps of CYFRA21-1 are as follows: (1) The three-electrode system of the electrochemical workstation was used for testing. All electrochemical measurements adopted the traditional three-electrode system: a saturated calomel electrode was used as the reference electrode, a platinum electrode was used as the counter electrode, and the prepared electrochemical immunosensor was used as the working electrode. The electrochemical measurements were carried out in PBS buffer with a pH of 7.38 by the DPV method. The parameters were set as follows: range and direction: -0.6 ~ 0.6 V, potential increment: 0.004 V, frequency: 15 Hz, amplitude: 0.05 V. (2) In 10 mL of PBS buffer solution with a pH of 6.0 to 9.2, the electrochemical signals generated by different concentrations of CYFRA21-1 standard solutions were detected by electrochemical method, and a working curve was drawn; (3) The CYFRA21-1 sample solution to be tested is used instead of the CYFRA21-1 standard solution for determination.