CN112326755B - Preparation method of photoelectric immunosensor for detection of lung cancer marker CYFRA21-1 based on HRP amplification - Google Patents

Abstract

The invention relates to a preparation method of an ultrasensitive photoelectrochemical immunoassay sensor for detecting lung cancer marker CYFRA21‑1 based on HRP amplification. The present invention uses BiVO ₄ /Ag ₃ VO4/SnS ₂ as the base material and irradiates with visible light to obtain photocurrent, and the combination of the three bases forms a heterojunction so that the photoelectric efficiency is greatly improved. The HRP-silica-CYFRA21-1 secondary antibody nanocomposite has a large steric hindrance, and after further HRP amplification, the electrode material is placed in 4-chloro-1-naphthol to generate an insoluble precipitate, which makes the electron supply The transfer of the body was triple hindered, which greatly changed the change of photocurrent, resulting in different photocurrents of antigens with different concentrations, thus realizing the detection of an ultra-wide range of CYFRA21‑1 antigen (100 ng/mL‑0.05 pg/ mL), with a detection limit of 0.01 pg/mL.

Description

Photoimmunosensor for detection of lung cancer marker CYFRA21-1 based on HRP amplification preparation method

technical field

The invention relates to a preparation method of an ultrasensitive photoelectrochemical immunoassay sensor for detecting lung cancer marker CYFRA21-1 based on HRP amplification. Specifically, BiVO ₄ /Ag ₃ VO4/SnS ₂ is used as the base material, and HRP-silicon dioxide composite As a secondary antibody marker, a photoelectrochemical sensor for detecting CYFRA21-1 antigen is prepared, which belongs to the technical field of new functional materials and biosensing detection.

Background technique

Lung cancer is one of the most common malignant tumors in today's society, with a high mortality rate and one of the main diseases that endanger people's lives. In 2011, there were approximately 650,000 new cases of lung cancer in my country, ranking first among male malignant tumors and second among females, second only to breast cancer. At present, lung cancer still ranks first in the mortality rate of malignant tumors, with 520,000 deaths per year due to lung cancer, ranking first in both male and female populations%5B1%5D. %20 The pathological type and tumor stage of lung cancer play a crucial role in its treatment plan and 5-year survival rate. Early diagnosis and treatment can significantly improve the prognosis and 5-year survival rate of patients, and at the same time improve the quality of life of patients. The use of biochemical means to detect tumor markers in human body fluids has certain advantages in the early diagnosis of tumors. CYFRA21-1 is a fragment of cytokeratin 19, which is composed of two monoclonal antibodies to cytokeratin 19 in the cell structure. It is one of the most sensitive tumor markers for screening lung cancer and has been widely used in the diagnosis and pathological classification of lung cancer. type and treatment evaluation. Therefore, it is very important to construct a rapid and sensitive analytical method for detecting CYFRA21-1. At present, there are many detection methods for CYFRA21-1, such as enzyme-linked immunoassay, fluorescence analysis, electrochemiluminescence analysis and so on. However, the operation of enzyme-linked immunoassay is complicated and cumbersome; the controllability of fluorescence analysis is poor, and the toxicity is high; the detection time of chemiluminescence immunoassay is long. The present invention designs a sandwich-type photoelectrochemical sensor. The sandwich-type sensor is sensitive in detection, high in accuracy and good in stability. The detection of the sandwich-type photoelectrochemical sensor designed in the present invention is simple and fast, stable and non-toxic, and has a detection limit of CYFRA21-1 Reached 0.01pg/mL.

Contents of the invention

One of the objectives of the present invention is to prepare BiVO ₄ /Ag ₃ VO4/SnS ₂ composite heterojunction material, which has better photoelectric response and high photoelectric conversion efficiency under visible light.

The second object of the present invention is to use HRP- _SiO2 as a secondary antibody marker, and simultaneously prepare HRP-tyramide complexes to amplify HRP. In the presence of HRP, 4-CN can be converted into 4-CD insoluble precipitates, so A large number of substances that hinder the transfer of electron donors are attached to the surface of the photoelectric substrate material, which greatly changes the photocurrent.

The third object of the present invention is to use BiVO ₄ /Ag ₃ VO4/SnS ₂ as the substrate, and use the HRP-SiO ₂ complex as the secondary antibody label to prepare a photoelectrochemical sensor with high sensitivity, good selectivity and fast detection speed. A sensor that achieves sensitive detection of the cancer marker CYFRA21-1 under visible light.

Technical scheme of the present invention is as follows:

1. The preparation method for detecting lung cancer marker CYFRA21-1 ultrasensitive photoelectrochemical immunoassay sensor based on HRP amplification is characterized in that, comprising the following steps:

(1) Preparation of bismuth vanadate

Take 0.06 ~ 0.15 g of bismuth nitrate pentahydrate and 0.046 ~ 0.06 g of sodium orthovanadate dissolved in 40 ~ 60 mL of water, after ultrasonication for 20 ~ 50 min, the mixed solution is transferred to the reaction kettle, and reacted at 160 ~ 180 °C for 8 ~ After 12 h, the reaction was cooled to room temperature, washed three times with absolute ethanol and ultrapure water, and dried in vacuum to obtain the product powder;

(2) Preparation of bismuth vanadate-silver vanadate composite

Dissolve 0.3 ~ 0.25 g of the prepared cerium oxide in 50 ~ 80 mL of water, then add 0.1 ~ 0.2 g of silver nitrate and stir for 30 ~ 50 min, then dissolve 0.044 ~ 0.05 g of sodium orthovanadate in 50 mL of water and stir for 2 h Then slowly add this solution into the above-mentioned bismuth vanadate solution, stir for 3~5h in the dark, wash with absolute ethanol and ultrapure water for 3 times, then vacuum dry at 40~60°C for 10~14 h;

(3) Preparation of tin sulfide

Take 0.3 ~ 0.5g of thioacetamide and 0.7 ~ 1.0g of tin tetrachloride pentahydrate dissolved in 50 ~ 100 mL of isopropanol solution, stir for 30 min, transfer to a 100 mL autoclave, and react at 180 °C Cool to room temperature for 18 to 24 hours, wash with ultrapure water and ethanol for 5 to 8 times, and finally dry the sample in a vacuum environment at 80°C for 12 hours to obtain the final product;

(4) Preparation of silica

Mix 60-80 mL of absolute ethanol with 2-5 mL of ultrapure water to form a transparent solution, then add 5-10 mL of tetraethyl silicate, and continue to add 10-30 mL at a rate of 2 mL/min with a mass fraction of 10 % ~ 30% ammonia water, stirred at 20 ~ 50 ° C for 3 ~ 6 h, centrifuged, washed with absolute ethanol and ultrapure water until neutral, and dried in vacuum at 30 ~ 50 ° C for 10 ~ 14 h, Silica material is obtained;

(5) Preparation of HRP-silica-CYFRA21-1 secondary antibody complex

Take 2 ~ 4 mL of APTES (3-aminopropyltriethoxysilane) and 1 ~ 2 g of the prepared silica and add it to 100 ~ 200 mL of anhydrous toluene. After the solution is sonicated for 20 ~ 30 min, the solution is placed in Stir at 80-100°C for 20-24 hours, then centrifuge and wash with ethanol and ultrapure water until neutral, and finally dry the product overnight at 40-60°C to obtain aminated silica powder;

Take 1 ~ 2 mL of 5 mg/mL aminated silica and add it to 0.5 ~ 1.0 mL of glutaraldehyde with a mass fraction of 2.5%, stir at room temperature for 6 hours, centrifuge and wash with PBS at pH 7.4 to remove the glutaraldehyde. Dialdehyde, and then the centrifuged product was dissolved in 1 mL of PBS with a pH of 7.4, and 200 to 400 μg/mL of CYFRA21-1 secondary antibody and 200 to 400 μL of HRP at a concentration of 1 to 5 mg/mL were added, Shake the solution vigorously at 37°C for 1 hour, and centrifuge to wash. Disperse the product obtained after washing in 2 mL of PBS solution with a pH of 7.4. At the same time, add 50-80 μL of BSA with a mass fraction of 1-3%. solution (PBS buffer pH 7.4), to block non-specific active sites, then vigorously shake at room temperature for 1 hour, centrifuge and wash, and finally disperse the product obtained after centrifugation in 5 mL, pH 7.4 in PBS solution and stored at 4 °C;

(6) Preparation of HRP - Tyramide

Take 1 ~ 3 mL of HRP solution and dissolve it in 3 ~ 5 mL of sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (pH=9.3), shake it for 5 min, and adjust the pH with 3 M hydrochloric acid solution 7.3 ~ 8.0, then add 30 ~ 50 mgNHS and 40 ~ 60mgEDC, shake for 45min, add 1.2 ~ 3mL of tyramine (5mg/mL), shake at room temperature for 12 ~ 18h, finally centrifuge at 4°C and use Wash twice with PBS, disperse in 5-8 mL PBS and store in a refrigerator at 4°C;

(7) Preparation of photoelectrochemical sensors

1) Clean the conductive glass with washing powder, acetone, ethanol and ultrapure water in sequence, and dry it with nitrogen;

2) Take 10 µL, 8 ~ 12 mg/mL of bismuth vanadate-silver vanadate complex and tin sulfide aqueous solution dropwise on the conductive surface of ITO conductive glass, and dry it under infrared lamp;

3) On the surface of the modified electrode, continue to drop 10 ~ 30 mg/mL 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10 ~ 30 mg /mL N-hydroxysuccinimide mixture 8 ~ 12 μL. Rinse the surface of the electrode with ultrapure water, and let it dry naturally at room temperature to a wet film state;

4) Add 10 μL, 5 ~ 20 μg/mL CYFRA21-1 capture antibody dropwise, wash with PBS, and let it dry naturally at room temperature to a wet film state;

5) Add 10 µL of bovine serum albumin solution with a mass fraction of 1 to 3% prepared in PBS buffer solution dropwise on the surface of the modified electrode, rinse the surface of the electrode with ultrapure water, and dry it in a refrigerator at 4 °C;

6) Add 10 µL, 0.01 pg/mL ~ 5 ng/mL of CYFRA21-1 antigen prepared in PBS buffer solution dropwise, rinse the electrode surface with ultrapure water, and dry it naturally in a refrigerator at 4 °C;

7) Add 10 μL, 5 ~ 20 μg/mL of CYFRA21-1 detection antibody labeled with HRP-silica, rinse the surface of the electrode with ultrapure water, and dry it in a 4 ℃ refrigerator until it is in a wet film state;

8) Add 3 µL H ₂ O ₂ , 7 µL HRP-tyramide dropwise and let it dry for 10 min;

9) Soak the material obtained above in a solution of 10 mM 4-1-naphthol and 0.15 mM H ₂ O ₂ for 10 to 30 min to obtain an electrode material;

2. The detection method of the photoelectrochemical sensor is as follows:

(1) The electrochemical workstation was used to test with a three-electrode system. The saturated calomel electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared ITO modified sensor was used as the working electrode. Tested in a PBS buffer solution with a pH of 5.0 to 8.0;

(2) The standard solution of CYFRA21-1 specific antigen prepared by serial dilution with PBS buffer solution was detected by the time-current method, the voltage was set at -0.1 ~ 0.1 V, the running time was 120 s, and the wavelength of the light source was a white mixed light source;

(3) After the electrode is placed, turn on the light every 20 s and continue to irradiate for 20 s, record the photocurrent, and draw the working curve;

(4) Replace the CYFRA21-1 antigen standard solution with the CYFRA21-1 antigen sample solution to be tested;

The linear detection range of this sensor for CYFRA21-1 antigen is 100 ng/mL - 0.05 pg/mL, and the detection limit is 0.01 pg/mL;

The chemical reagents required for the synthesis of materials were all purchased from local reagent stores without further processing.

Beneficial results of the present invention

(1) The present invention successfully synthesized a new BiVO ₄ /Ag ₃ VO4/SnS ₂ composite material with high photoelectric conversion efficiency, which solved the problem of low photoelectric conversion efficiency of pure BiVO ₄ and pure SnS ₂ ;

(2) HRP-SiO ₂ is used as a secondary antibody label, and the HRP-tyramide complex is prepared at the same time to allow HRP to amplify. In the presence of HRP, 4-CN can be converted into 4-CD insoluble precipitates, so in the optoelectronic substrate material A large number of substances that hinder the transfer of electron donors are attached to the surface, which greatly changes the photocurrent;

(3) Using BiVO ₄ /Ag ₃ VO4/SnS ₂ as the substrate and HRP-SiO ₂ complex as the secondary antibody label, a photoelectrochemical sensor with high sensitivity, good selectivity and fast detection speed was prepared, realizing the Sensitive detection of the cancer marker CYFRA21-1 under visible light;

(4) The photoelectrochemical sensor prepared by the present invention is used for the detection of CYFRA21-1 antigen. It has short response time, low detection limit, wide linear range and good stability, and can realize simple, fast, high sensitivity and high stability detection.

specific implementation plan

Embodiment 1 Preparation of photoelectrochemical sensor

(1) Preparation of bismuth vanadate

Dissolve 0.06 g of bismuth nitrate pentahydrate and 0.046 g of sodium orthovanadate in 40 mL of water. After ultrasonication for 20 min, the mixed solution is transferred to a reaction kettle and reacted at 160 ° C for 8 h. Wash with ultrapure water for 3 times, and vacuum dry to obtain product powder;

(2) Preparation of bismuth vanadate-silver vanadate composite

Dissolve 0.3 g of the prepared ceria in 50 mL of water, then add 0.1 g of silver nitrate and stir for 30 min, then dissolve 0.044 g of sodium orthovanadate in 50 mL of water and stir for 2 h, then slowly add the solution to the vanadic acid Bismuth solution, stirred for 3 h in the dark, washed with absolute ethanol and ultrapure water three times, and dried in vacuum at 40°C for 10 h;

(3) Preparation of tin sulfide

Dissolve 0.3 g of thioacetamide and 0.7 g of tin tetrachloride pentahydrate in 50 mL of isopropanol solution, stir for 30 min, transfer to a 100 mL autoclave, react at 180 °C for 18 h, cool to room temperature and use Wash with ultrapure water and ethanol for 5 times, and finally place the sample in a vacuum environment at 80°C for 12 h to obtain the final product;

(4) Preparation of silica

Mix 60 mL of absolute ethanol and 2 mL of ultrapure water to form a transparent solution, then add 5 mL of tetraethyl silicate, continue to add 10 mL of ammonia water with a mass fraction of 10% at a rate of 2 mL/min, and set the temperature at 20 °C After stirring at low temperature for 3 h, centrifuge, wash the product with absolute ethanol and ultrapure water until neutral, and dry it in vacuum at 30 °C for 10 h to obtain a silica material;

(5) Preparation of HRP-silica-CYFRA21-1 secondary antibody complex

Take 2 mL of APTES (3-aminopropyltriethoxysilane) and 1 g of the prepared silica and add it to 100 mL of anhydrous toluene. After the solution is sonicated for 20 min, the solution is stirred at 80°C for 20 h , then centrifuged and washed with ethanol and ultrapure water until neutral, and finally the product was dried overnight at 40°C to obtain aminated silica powder;

Add 1 mL of 5 mg/mL aminated silica to 0.5 mL of glutaraldehyde with a mass fraction of 2.5%, stir at room temperature for 6 hours, centrifuge and wash with PBS at pH 7.4 to remove glutaraldehyde, then Dissolve the centrifuged product in 1 mL of PBS with a pH of 7.4, add 200 μg/mL of CYFRA21-1 secondary antibody and 200 μL of 1 mg/mL HRP, shake the solution vigorously at 37°C for 1 hours, and centrifuged for washing, the product obtained after washing was dispersed in 2 mL of PBS solution with a pH of 7.4, and 50 μL of BSA solution with a mass fraction of 1% (prepared in PBS buffer with a pH of 7.4) was added at the same time to To block the non-specific active sites, shake vigorously at room temperature for 1 hour, centrifuge and wash, and finally disperse the product obtained after centrifugation in 5 mL of PBS solution with pH 7.4, and store at 4 °C;

(6) Preparation of HRP - Tyramide

Dissolve 1 mL of HRP solution in 3 mL of sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (pH=9.3) and shake for 5 min, adjust the pH to 7.3 with 3 M hydrochloric acid solution, and then add 30 mg NHS and 40 mg EDC, add 1.2 mL of tyramine (5 mg/mL) after shaking for 45 min, shake at room temperature for 12 h, finally centrifuge at 4 °C and wash twice with PBS, disperse in 5 mL of PBS and place at 4 Store in refrigerator;

(7) Preparation of photoelectrochemical sensors

1) Clean the conductive glass with washing powder, acetone, ethanol and ultrapure water in sequence, and dry it with nitrogen;

2) Add 10 µL, 8 mg/mL bismuth vanadate-silver vanadate complex and tin sulfide aqueous solution dropwise to the conductive surface of the ITO conductive glass, and dry it under the infrared lamp;

3) On the surface of the modified electrode, continue to drop 10 mg/mL 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10 mg/mL N- 8 μL of the mixture of hydroxysuccinimide. Rinse the surface of the electrode with ultrapure water, and let it dry naturally at room temperature to a wet film state;

4) Add 10 μL, 5 μg/mL CYFRA21-1 capture antibody dropwise, wash with PBS, and let it dry naturally at room temperature to a wet film state;

5) Add 10 µL of bovine serum albumin solution with a mass fraction of 1 to 3% prepared in PBS buffer solution dropwise on the surface of the modified electrode, rinse the surface of the electrode with ultrapure water, and dry it in a refrigerator at 4 °C;

6) Add 10 µL, 0.01 pg/mL of CYFRA21-1 antigen prepared in PBS buffer solution dropwise, rinse the surface of the electrode with ultrapure water, and dry it naturally in a refrigerator at 4 °C;

7) Add 10 µL, 5 µg/mL CYFRA21-1 detection antibody labeled with HRP-silica, rinse the surface of the electrode with ultrapure water, and dry it in a 4°C refrigerator until it is in a wet film state;

8) Add 3 µL H ₂ O ₂ , 7 µL HRP-tyramide dropwise and let it dry for 10 min;

9) Soak the material obtained above in a solution of 10 mM 4-1-naphthol and 0.15 mM H ₂ O ₂ for 10 min to obtain an electrode material;

2. The detection method of the photoelectrochemical sensor is as follows:

(1) The electrochemical workstation was used to test with a three-electrode system. The saturated calomel electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared ITO modified sensor was used as the working electrode. 5.0 in PBS buffer solution for testing;

(2) The standard solution of CYFRA21-1 specific antigen prepared by serial dilution with PBS buffer solution was detected by the time-current method, the voltage was set at -0.1 V, the running time was 120 s, and the wavelength of the light source was a white mixed light source;

(3) After the electrode is placed, turn on the light every 20 s and continue to irradiate for 20 s, record the photocurrent, and draw the working curve;

(4) Replace the CYFRA21-1 antigen standard solution with the CYFRA21-1 antigen sample solution to be tested for detection.

Example 2 Preparation of photoelectrochemical sensor

(1) Preparation of ceria

(1) Preparation of bismuth vanadate

Dissolve 0.07 g of bismuth nitrate pentahydrate and 0.05 g of sodium orthovanadate in 50 mL of water. After ultrasonication for 30 min, the mixed solution is transferred to a reaction kettle and reacted at 160 ° C for 10 h. Wash with ultrapure water for 3 times, and vacuum dry to obtain product powder;

(2) Preparation of bismuth vanadate-silver vanadate composite

Dissolve 0.35 g of the prepared ceria in 80 mL of water, then add 0.1 g of silver nitrate and stir for 40 min, then dissolve 0.044 g of sodium orthovanadate in 50 mL of water and stir for 2 h, then slowly add the solution to the vanadic acid Bismuth solution, stirred for 3 h in the dark, washed with absolute ethanol and ultrapure water three times, and dried in vacuum at 40°C for 10 h;

(3) Preparation of tin sulfide

Dissolve 0.4 g of thioacetamide and 0.75 g of tin tetrachloride pentahydrate in 50 mL of isopropanol solution, stir for 30 min, transfer to a 100 mL autoclave, react at 180 °C for 20 h, cool to room temperature and use Wash with ultrapure water and ethanol for 5 times, and finally place the sample in a vacuum environment at 80°C for 12 h to obtain the final product;

(4) Preparation of silica

Mix 60 mL of absolute ethanol and 2.5 mL of ultrapure water to form a transparent solution, then add 5 mL of tetraethyl silicate, continue to add 10 mL of ammonia water with a mass fraction of 15% at a rate of 2 mL/min, and set the temperature at 20 °C After stirring at low temperature for 3 h, centrifuge, wash the product with absolute ethanol and ultrapure water until neutral, and dry it in vacuum at 30 °C for 10 h to obtain a silica material;

(5) Preparation of HRP-silica-CYFRA21-1 secondary antibody complex

Take 3 mL of APTES (3-aminopropyltriethoxysilane) and 1 g of prepared silica and add it to 100 mL of anhydrous toluene. After the solution is sonicated for 20 min, the solution is stirred at 80°C for 20 h , then centrifuged and washed with ethanol and ultrapure water until neutral, and finally the product was dried overnight at 40°C to obtain aminated silica powder;

Add 1 mL of 5 mg/mL aminated silica to 0.5 mL of glutaraldehyde with a mass fraction of 2.5%, stir at room temperature for 6 hours, centrifuge and wash with PBS at pH 7.4 to remove glutaraldehyde, then Dissolve the centrifuged product in 1 mL of PBS with a pH of 7.4, add 20 μg/mL of CYFRA21-1 secondary antibody and 200 μL of 1 mg/mL HRP, shake the solution vigorously at 37°C for 1 hours, and centrifuged for washing, the product obtained after washing was dispersed in 2 mL of PBS solution with a pH of 7.4, and 50 μL of BSA solution with a mass fraction of 1% (prepared in PBS buffer with a pH of 7.4) was added at the same time to To block the non-specific active sites, shake vigorously at room temperature for 1 hour, centrifuge and wash, and finally disperse the product obtained after centrifugation in 5 mL of PBS solution with pH 7.4, and store at 4 °C;

(6) Preparation of HRP - Tyramide

Dissolve 1 mL of HRP solution in 4 mL of sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (pH=9.3) and shake for 5 min, adjust the pH to 7.3 with 3 M hydrochloric acid solution, and then add 30 mg NHS and 40 mg EDC, add 1.5 mL of tyramine (5 mg/mL) after shaking for 45 min, shake at room temperature for 14 h, finally centrifuge at 4 °C and wash twice with PBS, disperse in 5 mL of PBS and place at 4 Store in refrigerator;

(7) Preparation of photoelectrochemical sensors

1) Clean the conductive glass with washing powder, acetone, ethanol and ultrapure water in sequence, and dry it with nitrogen;

2) Add 10 µL, 8 mg/mL bismuth vanadate-silver vanadate complex and tin sulfide aqueous solution dropwise to the conductive surface of the ITO conductive glass, and dry it under the infrared lamp;

3) On the surface of the modified electrode, continue to drop 15 mg/mL 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10 mg/mL N- 8 μL of the mixture of hydroxysuccinimide. Rinse the surface of the electrode with ultrapure water, and let it dry naturally at room temperature to a wet film state;

4) Add 10 μL, 5 μg/mL CYFRA21-1 capture antibody dropwise, wash with PBS, and let it dry naturally at room temperature to a wet film state;

5) Add 10 µL of bovine serum albumin solution with a mass fraction of 1 to 3% prepared in PBS buffer solution dropwise on the surface of the modified electrode, rinse the surface of the electrode with ultrapure water, and dry it in a refrigerator at 4 °C;

6) Add 10 µL, 0.02 pg/mL of CYFRA21-1 antigen prepared in PBS buffer solution dropwise, rinse the surface of the electrode with ultrapure water, and dry it naturally in a refrigerator at 4 °C;

7) Add 10 µL, 5 µg/mL CYFRA21-1 detection antibody labeled with HRP-silica, rinse the surface of the electrode with ultrapure water, and dry it in a 4°C refrigerator until it is in a wet film state;

8) Add 3 µL H ₂ O ₂ , 7 µL HRP-tyramide dropwise and let it dry for 10 min;

9) Soak the material obtained above in a solution of 10 mM 4-1-naphthol and 0.2 mM H ₂ O ₂ for 20 min to obtain an electrode material;

2. The detection method of the photoelectrochemical sensor is as follows:

(1) The electrochemical workstation was used to test with a three-electrode system. The saturated calomel electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared ITO modified sensor was used as the working electrode. 5.0 in PBS buffer solution for testing;

(2) The standard solution of CYFRA21-1 specific antigen prepared by serial dilution with PBS buffer solution was detected by the time-current method, the voltage was set to 0 V, the running time was 120 s, and the wavelength of the light source was a white mixed light source;

(3) After the electrode is placed, turn on the light every 20 s and continue to irradiate for 20 s, record the photocurrent, and draw the working curve;

(4) Replace the CYFRA21-1 antigen standard solution with the CYFRA21-1 antigen sample solution to be tested for detection.

Example 3 Detection of CYFRA21-1 Antigen

(1) The electrochemical workstation was used to test with a three-electrode system. The saturated calomel electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared ITO modified sensor was used as the working electrode. 5.0 in PBS buffer solution for testing;

(2) The standard solution of CYFRA21-1 antigen prepared by serial dilution with PBS buffer solution was detected by time-current method, the voltage was set to 0 V, the running time was 120 s, and the wavelength of the light source was 400 nm;

(3) After the electrode is placed, turn on the light every 20 s and continue to irradiate for 20 s, record the photocurrent, and draw the working curve;

(4) Replace the CYFRA21-1 antigen standard solution with the CYFRA21-1 antigen sample solution to be tested for detection.

Example 4 Detection of CYFRA21-1 Antigen

(1) The electrochemical workstation was used to test with a three-electrode system. The saturated calomel electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared ITO modified sensor was used as the working electrode. Tested in PBS buffer solution of 7.4;

(2) The standard solution of CYFRA21-1 antigen diluted with PBS buffer solution was detected by time-current method, the voltage was set at 0 V, the running time was 120 s, and the wavelength of the light source was 430 nm;

(3) After the electrode is placed, turn on the light every 20 s and continue to irradiate for 20 s, record the photocurrent, and draw the working curve;

(4) Replace the CYFRA21-1 antigen standard solution with the CYFRA21-1 antigen sample solution to be tested for detection.

Claims (2)

1. The preparation method for detecting lung cancer marker CYFRA21-1 photoelectric immunosensor based on HRP amplification, is characterized in that, comprises the following steps: (1) Preparation of bismuth vanadate Take 0.06 ~ 0.15 g of bismuth nitrate pentahydrate and 0.046 ~ 0.06 g of sodium orthovanadate dissolved in 40 ~ 60 mL of water, after ultrasonication for 20 ~ 50 min, the mixed solution is transferred to the reaction kettle, and reacted at 160 ~ 180 °C for 8 ~ After 12 h, the reaction was cooled to room temperature, washed three times with absolute ethanol and ultrapure water, and dried in vacuum to obtain the product powder; (2) Preparation of bismuth vanadate-silver vanadate composite Dissolve 0.3 ~ 0.25 g of the prepared ceria in 50 ~ 80 mL of water, then add 0.1 ~ 0.2 g of silver nitrate and stir for 30 ~ 50 min, then dissolve 0.044 ~ 0.05 g of sodium orthovanadate in 50 mL of water and stir for 2 h Then slowly add this solution into the above-mentioned bismuth vanadate solution, stir for 3-5 hours in the dark, wash with absolute ethanol and ultrapure water for 3 times, and then vacuum-dry at 40-60°C for 10-14 hours; (3) Preparation of tin sulfide Take 0.3 ~ 0.5g of thioacetamide and 0.7 ~ 1.0g of tin tetrachloride pentahydrate dissolved in 50 ~ 100 mL of isopropanol solution, stir for 30 min, transfer to a 100 mL autoclave, and react at 180 °C Cool to room temperature for 18 to 24 hours, wash with ultrapure water and ethanol for 5 to 8 times, and finally dry the sample in a vacuum environment at 80°C for 12 hours to obtain the final product; (4) Preparation of silica Mix 60-80 mL of absolute ethanol with 2-5 mL of ultrapure water to form a transparent solution, then add 5-10 mL of tetraethyl silicate, and continue to add 10-30 mL at a rate of 2 mL/min with a mass fraction of 10 % ~ 30% ammonia water, stirred at 20 ~ 50 ° C for 3 ~ 6 h, centrifuged, washed with absolute ethanol and ultrapure water until neutral, and dried in vacuum at 30 ~ 50 ° C for 10 ~ 14 h, Silica material is obtained; (5) Preparation of HRP-silica-CYFRA21-1 secondary antibody complex Take 2 ~ 4 mL of APTES (3-aminopropyltriethoxysilane) and 1 ~ 2 g of the prepared silica and add it to 100 ~ 200 mL of anhydrous toluene. After the solution is sonicated for 20 ~ 30 min, the solution is placed in Stir at 80-100°C for 20-24 hours, then centrifuge and wash with ethanol and ultrapure water until neutral, and finally dry the product overnight at 40-60°C to obtain aminated silica powder; Take 1 ~ 2 mL of 5 mg/mL aminated silica and add it to 0.5 ~ 1.0 mL of glutaraldehyde with a mass fraction of 2.5%, stir at room temperature for 6 hours, centrifuge and wash with PBS at pH 7.4 to remove the glutaraldehyde. Dialdehyde, and then the centrifuged product was dissolved in 1 mL of PBS with a pH of 7.4, and 200 to 400 μg/mL of CYFRA21-1 secondary antibody and 200 to 400 μL of HRP at a concentration of 1 to 5 mg/mL were added, Shake the solution vigorously at 37°C for 1 hour, and centrifuge to wash. Disperse the product obtained after washing in 2 mL of PBS solution with a pH of 7.4. At the same time, add 50-80 μL of BSA with a mass fraction of 1-3%. Solution, in order to block non-specific active sites, then shake vigorously at room temperature for 1 hour, centrifuge and wash, and finally disperse the product obtained after centrifugation in 5 mL of PBS solution with pH 7.4, and incubate at 4 °C Save under; (6) Preparation of HRP - Tyramide Take 1 ~ 3 mL of HRP solution dissolved in 3 ~ 5 mL of sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate, shake for 5 min, adjust the pH to 7.3 ~ 8.0 with 3 M hydrochloric acid solution, Then add 30 ~ 50 mg NHS and 40 ~ 60 mg EDC, shake for 45 min, add 1.2 ~ 3 mL of tyramine, shake at room temperature for 12 ~ 18 h, finally centrifuge at 4 °C and wash twice with PBS, disperse in 5 ~ 8 mL PBS and store in a 4°C refrigerator; (7) Preparation of photoelectrochemical sensors 1) Clean the conductive glass with washing powder, acetone, ethanol and ultrapure water in sequence, and dry it with nitrogen; 2) Take 10 µL, 8 ~ 12 mg/mL of bismuth vanadate-silver vanadate complex and tin sulfide aqueous solution dropwise on the conductive surface of ITO conductive glass, and dry it under infrared lamp; 3) On the surface of the modified electrode, continue to drop 10 ~ 30 mg/mL 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10 ~ 30 mg /mL N-hydroxysuccinimide mixture 8 ~ 12 μL; rinse the electrode surface with ultrapure water, and let it dry naturally at room temperature until it is in a wet film state; 4) Add 10 μL, 5 ~ 20 μg/mL CYFRA21-1 capture antibody dropwise, wash with PBS, and let it dry naturally at room temperature to a wet film state; 5) Add 10 µL of bovine serum albumin solution with a mass fraction of 1 to 3% prepared in PBS buffer solution dropwise on the surface of the modified electrode, rinse the surface of the electrode with ultrapure water, and dry it in a refrigerator at 4 °C; 6) Add 10 µL, 0.01 pg/mL ~ 5 ng/mL of CYFRA21-1 antigen prepared in PBS buffer solution dropwise, rinse the surface of the electrode with ultrapure water, and dry it naturally in a refrigerator at 4 °C; 7) Add 10 μL, 5 ~ 20 μg/mL of CYFRA21-1 detection antibody labeled with HRP-silica, rinse the surface of the electrode with ultrapure water, and dry it in a 4 ℃ refrigerator until it is in a wet film state; 8) Add 3 µL H ₂ O ₂ , 7 µL HRP-tyramide dropwise and let it dry for 10 min; 9) Soak the material obtained above in a solution of 10 mM 4-1-naphthol and 0.15 mM H ₂ O ₂ for 10 to 30 min to obtain an electrode material. 2. the detection method of the photoelectrochemical sensor prepared as claimed in claim 1, is characterized in that, step is as follows: (1) The electrochemical workstation was used to test with a three-electrode system. The saturated calomel electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared ITO modified sensor was used as the working electrode. Tested in a PBS buffer solution with a pH of 5.0 to 8.0; (2) The standard solution of CYFRA21-1 specific antigen prepared by serial dilution with PBS buffer solution was detected by the time-current method, the voltage was set at -0.1 ~ 0.1 V, the running time was 120 s, and the wavelength of the light source was a white mixed light source; (3) After the electrode is placed, turn on the light every 20 s and continue to irradiate for 20 s, record the photocurrent, and draw the working curve; (4) Replace the CYFRA21-1 antigen standard solution with the CYFRA21-1 antigen sample solution to be tested for detection.