Preparation Method and Application of a Multiple Inhibitory Quenching-Type Photoelectrochemical Sensor

Abstract

The present invention belongs to the technical field of immunoassay and biosensing, and designs a preparation method and application of a multiple inhibition quenching type photoelectrochemical sensor; the present invention adopts photoelectrochemical technology, and by designing and synthesizing MgFe2O4@CdS nanocomposite materials as photoelectric materials, with Fe 3+ doped polydopamine microspheres SMNP-Fe 3+ incubated with the detection antibody as a secondary antibody marker to construct a photoelectrochemical sensor with a multiple-mode quenching function; the sensor developed based on this method is applied to the sensitive detection of the lung cancer marker CYFRA21-1, and has the advantages of high sensitivity, strong specificity, rapid response, etc., and its detection limit is as low as 0.16 pg / mL, which has important scientific significance and application value for the early diagnosis and disease monitoring of lung cancer.

Description

Technical Field

[0001] The present invention belongs to the technical fields of nano-functional materials, immunoassay, and biosensing technology, and relates to a preparation method and application of a multiple inhibition quenching type photoelectrochemical sensor, belonging to the fields of photoelectrochemistry and immunosensing. Background Art

[0002] Cytokeratin 19 fragment CYFRA21-1 is a tumor marker with high diagnostic value in lung cancer. Therefore, constructing a sensitive and effective detection method for CYFRA21-1 to achieve sensitive and efficient in vitro detection of lung cancer markers is of great significance for the prevention and early diagnosis of lung cancer, achieving "early detection, early diagnosis, and early treatment", and improving the survival rate of patients.

[0003] A photoelectric biosensor is a signal conversion device that converts specific biological recognition into visualizable current, and has increasingly important value in the fields of biomedicine, environmental detection, food safety, and medical research. The present invention adopts photoelectrochemical technology, and by designing and synthesizing a nano-composite material of photoelectric material MgFe2O4@CdS as the photoelectric material, with SMNP-Fe 3+ as the secondary antibody label, a photoelectrochemical sensor with a multiple-mode quenching function is constructed. This method has the advantages of simple preparation and low cost, making up for the disadvantage that the sensitivity of the quenching type sensor is weaker than that of the label-free photoelectric sensor. The sensor developed based on this method is applied to the sensitive detection of the lung cancer marker CYFRA21-1, and has the advantages of high sensitivity, strong specificity, rapid response, and portability, and has important scientific significance and application value for the early diagnosis and disease monitoring of lung cancer. Summary of the Invention

[0004] The present invention provides a preparation method and application of a multiple inhibition quenching type photoelectrochemical sensor, realizing the highly sensitive detection of CYFRA21-1.

[0005] One of the purposes of the present invention is to provide a preparation method of a multiple inhibition quenching type photoelectrochemical sensor.

[0006] Another purpose of the present invention is to apply the prepared multiple inhibition quenching type photoelectrochemical sensor to the highly sensitive and specific detection of CYFRA21-1.

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

[0008] 1. A preparation method of a multiple inhibition quenching type photoelectrochemical sensor, characterized by comprising the following steps:

[0009] (1) The indium tin oxide (ITO) conductive glass electrodes cut into a size of 2.0 cm × 0.7 cm were ultrasonically cleaned in acetone, ultrapure water, absolute ethanol, and ultrapure water for 1 h in sequence, and then dried with nitrogen gas.

[0010] (2) 6 μL of a MgFe2O4 dispersion with a concentration of 2.0 - 6.0 mg / mL was dropped onto the conductive surface of the ITO electrode and air-dried at room temperature.

[0011] (3) The ITO electrode was then dipped into 0.8 mol / L Cd(NO3)2 and 0.1 mol / L Na2S solutions for 30 s in sequence, repeated 2 - 10 times. After drying at room temperature, CdS was formed on the surface of MgFe2O4 and combined fully, and the formed MgFe2O4@CdS was used as the substrate material.

[0012] (4) The ITO electrode was further immersed in a 1 - 5 mmol / L mercaptoacetic acid solution for 30 min to introduce carboxyl groups on the surface of MgFe2O4@CdS, and then rinsed thoroughly with ultrapure water.

[0013] (5) 3 μL of a 1:1 mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide with a concentration of 0.01 mol / L was dropped onto the surface of the above ITO electrode and activated at room temperature for 1 h. Then, 6 μL of a capture antibody Ab1 solution with a concentration of 10 μg / mL was further dropped and incubated at room temperature for 1 h, and then rinsed with a phosphate buffer solution (PBS) with pH = 7.4.

[0014] (6) 3 μL of a bovine serum albumin solution (BSA) with a mass fraction of 0.01 - 1% was dropped to block the non-specific active sites on the electrode surface. After incubating at room temperature for 30 min, it was rinsed with a PBS solution with pH = 7.4. (7) Then, 6 μL of a series of CYFRA21-1 solutions with different concentrations from 10 pg / mL to 100 ng / mL was further dropped. After rinsing the electrode surface with ultrapure water and incubating at 37 °C for 0.5 - 2 h, the electrode surface was rinsed with a PBS solution with pH = 7.4.

[0015] (8) 6 μL of a dispersion of detection antibody Ab2-labeled SMNP-Fe with a concentration of 2.0 - 4.0 mg / mL was dropped, and the electrode surface was rinsed with a PBS solution with pH = 7.4 to prepare a multiple inhibition quenching type photoelectrochemical sensor. 2. The dispersion of detection antibody Ab2-labeled SMNP-Fe 3+ is characterized in that the preparation steps are as follows: 3+

[0016] Dissolve 30 - 60 mg of dopamine hydrochloride and 4.0 - 8.0 mg of ferric chloride hexahydrate in 130 mL of ultrapure water. After stirring for 1 h, quickly add 20 mL of an aqueous solution containing 300 - 600 mg of tris(hydroxymethyl)aminomethane, and continue stirring for 1 - 3 h; perform centrifugal separation, wash the precipitate three times with ultrapure water to obtain SMNP-Fe 3+ nano-microspheres, disperse them in 1.0 mL of ultrapure water for standby; take 0.5 - 2.0 mL of SMNP-Fe 3+ at a concentration of 18 mg / mL and 1 - 10 mL of Ab2 at a concentration of 10 μg / mL and add them to a centrifuge tube. After thoroughly mixing the solution, incubate it with shaking at 4 °C for 8 - 12 h, perform centrifugal washing, and disperse it evenly in 0.5 - 10 mL of PBS buffer solution with pH = 7.4 to obtain a SMNP-Fe 3+ -Ab2 dispersion, store it in a refrigerator at 4 °C for standby.

[0017] 3. The MgFe2O4 dispersion liquid as described above is characterized in that the preparation steps are as follows:

[0018] Mix 40 - 80 mL of an ethylene glycol solution containing 0.1 g of polyvinylpyrrolidone with 1 - 20 mL of an ethylene glycol solution containing 0.81 g of Fe(NO3)2·6H2O and 0.64 g of Mg(NO3)2·6H2O and stir evenly. Subsequently, add 0.1 - 2.0 g of polyethylene glycol and 1.0 - 6.0 g of sodium acetate, and ultrasonically mix for 1 h; transfer the above solution to a high-pressure reaction kettle and react at 200 °C for 24 h; after cooling to room temperature, wash it with ethanol and ultrapure water by magnetic separation, and dry it in a vacuum drying oven for 12 h. Ultrasonically disperse the ground magnesium ferrite nanomaterial in ultrapure water to prepare a MgFe2O4 dispersion liquid for standby.

[0019] 4. The detection for the concentration of the lung cancer biomarker CYFRA21-1 as described above is characterized in that the operation steps are as follows: (1) Use an electrochemical workstation, with a saturated calomel electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared sensor as the working electrode to form a three-electrode system, and perform tests in a PBS bottom solution with pH = 7.4 containing 0.15 mol / L of ascorbic acid;

[0020] (2) Perform detection by the time - current method, with the light source being the full wavelength and the set bias voltage being 0 V;

[0021] (3) After placing the electrode, turn on the light and irradiate continuously for 20 s every 20 s, and detect the photocurrent intensity;

[0022] (4) Use standard solutions of CYFRA21-1 with different concentrations for determination, record the changes in the photocurrent values corresponding to the standard solutions of CYFRA21-1 with different concentrations, and plot a working curve;

[0023] (5) Test the photoelectrochemical sensor for incubating the actual sample of CYFRA21-1 with unknown concentration to obtain the corresponding signal intensity, and then calculate the concentration of CYFRA21-1 in the reagent sample according to the working curve.

[0024] All raw materials used in the present invention can be purchased from chemical reagent companies or biopharmaceutical companies.

[0025] Advantages of the present invention

[0026] (1) The present invention first uses the prepared SMNP-Fe 3+ as a detection antibody marker with multiple inhibition quenching functions; this marker can not only undergo redox reactions with ascorbic acid in the bottom solution, hinder the effective separation of electron-hole pairs in the substrate material, and at the same time compete with the substrate material for light absorption rate, playing a dual inhibition role and increasing the change value of photocurrent; SMNPs-Fe 3+ has high biocompatibility, realizes the fixation of antibodies, and meets the requirements of trace analysis;

[0027] (2) The present invention first constructs a multiple quenching type sensing system for the sensitive detection of CYFRA21-1 based on MgFe2O4@CdS with high photoelectric conversion efficiency as the base material. Its linear range is 0.5 pg / mL to 50 ng / mL, and the detection limit is 0.16 pg / mL, providing a new analytical method for the rapid detection of CYFRA21-1 and a strong theoretical and technical basis for the popular detection of tumor markers. Specific embodiments

[0028] The following combines specific embodiments to further elaborate the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0029] Embodiment 1 A preparation method of a photoelectrochemical sensor with multiple mode quenching functions, characterized by including the following steps:

[0030] (1) Ultrasonically clean the indium tin oxide (ITO) conductive glass electrode cut into a size of 2.0 cm × 0.7 cm in acetone, ultrapure water, absolute ethanol, and ultrapure water for 1 h in sequence, and dry it with nitrogen;

[0031] (2) Take 6 μL of a 2.0 mg / mL MgFe2O4 dispersion solution and drop it onto the conductive surface of the ITO electrode, and dry it at room temperature;

[0032] (3) Continuously dip the ITO electrode into 0.8 mol / L Cd(NO3)2 and 0.1 mol / L Na2S solutions for 30 s each, repeat 2 times, and dry at room temperature. CdS is formed and firmly combined on the surface of MgFe2O4 to form MgFe2O4@CdS as the substrate material;

[0033] (4) Continuously immerse the ITO electrode into a 1 mmol / L mercaptoacetic acid solution for 30 min to introduce carboxyl groups onto the surface of MgFe2O4@CdS, and then rinse thoroughly with ultrapure water;

[0034] (5) Drop 3 μL of a 1:1 mixed solution of 0.01 mol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide onto the surface of the above ITO electrode and activate at room temperature for 1 h; then continue to drop-coat 6 μL of a 10 μg / mL capture antibody Ab1 solution, incubate at room temperature for 1 h, and then rinse with a phosphate buffer solution PBS with pH = 7.4;

[0035] (6) Drop 3 μL of a 0.01% bovine serum albumin solution BSA to block the non-specific active sites on the electrode surface, incubate at room temperature for 30 min, and then rinse with a PBS solution with pH = 7.4;

[0036] (7) Continue to drop 6 μL of a series of different concentrations of CYFRA21-1 solutions ranging from 10 pg / mL to 100 ng / mL, rinse the surface of the electrode with ultrapure water, incubate at 37 °C for 0.5 h, and then rinse the surface of the electrode with a PBS with pH = 7.4;

[0037] (8) Drop 6 μL of a dispersion of detection antibody Ab2-labeled SMNP-Fe 3+ with a concentration of 2.0 mg / mL, rinse the surface of the electrode with a PBS with pH = 7.4, and a multiple inhibition quenching type photoelectrochemical sensor is prepared. Example 2 A method for preparing a multiple inhibition quenching type photoelectrochemical sensor, characterized by comprising the following steps:

[0038] (1) Ultrasonically clean an indium tin oxide ITO conductive glass electrode cut into a size of 2.0 cm × 0.7 cm in acetone, ultrapure water, absolute ethanol, and ultrapure water for 1 h each, and then dry with nitrogen;

[0039] (2) Take 6 μL of a 4.0 mg / mL MgFe2O4 dispersion and drop it onto the conductive surface of the ITO electrode, and dry at room temperature;

[0040] (3) Continuously dip the ITO electrode into 0.8 mol / L Cd(NO3)2 and 0.1 mol / L Na2S solutions for 30 s each, repeat 5 times, and after drying at room temperature, CdS is formed on the surface of MgFe2O4 and binds well, forming MgFe2O4@CdS as the substrate material;

[0041] (4) Continuously immerse the ITO electrode into a 2.5 mmol / L mercaptoacetic acid solution for 30 min to introduce carboxyl groups on the surface of MgFe2O4@CdS, and rinse thoroughly with ultrapure water;

[0042] (5) Drop 3 μL of a 1:1 mixed solution of 0.01 mol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide onto the surface of the above ITO electrode and activate at room temperature for 1 h; then continue to drop-coat 6 μL of a 10 μg / mL capture antibody Ab1 solution, incubate at room temperature for 1 h, and rinse with phosphate buffer solution PBS with pH = 7.4;

[0043] (6) Drop 3 μL of a 0.5% bovine serum albumin solution BSA to block the non-specific active sites on the electrode surface, incubate at room temperature for 30 min, and rinse with PBS solution with pH = 7.4;

[0044] (7) Continue to drop 6 μL of a series of different concentrations of CYFRA21-1 solutions from 10 pg / mL to 100 ng / mL, rinse the electrode surface with ultrapure water, incubate at 37 °C for 1 h, and then rinse the electrode surface with PBS with pH = 7.4;

[0045] (8) Drop 6 μL of a dispersion of detection antibody Ab2-labeled SMNP-Fe 3+ with a concentration of 3.0 mg / mL, rinse the electrode surface with PBS with pH = 7.4 to obtain a multiple inhibition quenching type photoelectrochemical sensor. Example 3 A method for preparing a multiple inhibition quenching type photoelectrochemical sensor, characterized by comprising the following steps:

[0046] (1) Ultrasonically clean an indium tin oxide ITO conductive glass electrode cut into a size of 2.0 cm × 0.7 cm in acetone, ultrapure water, absolute ethanol, and ultrapure water for 1 h each, and dry with nitrogen;

[0047] (2) Take 6 μL of a 6.0 mg / mL MgFe2O4 dispersion and drop it onto the conductive surface of the ITO electrode, and dry at room temperature;

[0048] (3) Continuously dip the ITO electrode into 0.8 mol / L Cd(NO3)2 and 0.1 mol / L Na2S solutions for 30 s each, repeat 10 times, and after drying at room temperature, CdS is formed on the surface of MgFe2O4 and binds well, forming MgFe2O4@CdS as the substrate material;

[0049] (4) Continuously immerse the ITO electrode into a 5 mmol / L mercaptoacetic acid solution for 30 min to introduce carboxyl groups on the surface of MgFe2O4@CdS, and then rinse it thoroughly with ultrapure water;

[0050] (5) Drop 3 μL of a 1:1 mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide with a concentration of 0.01 mol / L onto the surface of the above ITO electrode and activate it at room temperature for 1 h; then continue to drop-coat 6 μL of a capture antibody Ab1 solution with a concentration of 10 μg / mL, incubate at room temperature for 1 h, and then rinse with phosphate buffer solution PBS with pH = 7.4;

[0051] (6) Drop 3 μL of a 1% bovine serum albumin solution BSA to block the non-specific active sites on the electrode surface, incubate at room temperature for 30 min, and then rinse with PBS solution with pH = 7.4;

[0052] (7) Continue to drop 6 μL of a series of different concentrations of CYFRA21-1 solutions from 10 pg / mL to 100 ng / mL, rinse the electrode surface with ultrapure water, incubate at 37 °C for 2 h, and then rinse the electrode surface with PBS with pH = 7.4;

[0053] (8) Drop 6 μL of a dispersion of detection antibody Ab2-labeled SMNP-Fe 3+ with a concentration of 4.0 mg / mL, rinse the electrode surface with PBS with pH = 7.4, and a multiplex inhibition quenching type photoelectrochemical sensor is prepared.

[0054] Example 4 Dispersion of detection antibody Ab2-labeled SMNP-Fe 3+ It is characterized in that the preparation steps are as follows:

[0055] Dissolve 30 mg of dopamine hydrochloride and 4.0 mg of ferric chloride hexahydrate in 130 mL of ultrapure water, stir for 1 h, then quickly add 20 mL of an aqueous solution containing 300 mg of tris(hydroxymethyl)aminomethane, and continue to stir for 1 h; perform centrifugal separation, wash the precipitate three times with ultrapure water to obtain SMNP-Fe 3+ nanospheres, disperse them in 1.0 mL of ultrapure water for standby; take 0.5 mL of SMNP-Fe with a concentration of 18 mg / mL 3+Add 1 mL of 10 μg / mL Ab2 to a centrifuge tube. After thoroughly mixing the solution, incubate it with shaking at 4°C for 8 h. Then, centrifuge and wash it, and disperse it evenly in 0.5 mL of PBS buffer solution with pH = 7.4 to obtain SMNP-Fe 3+ -Ab2 dispersion, which is stored in a refrigerator at 4°C for later use.

[0056] Example 5 Detection of SMNP-Fe labeled with antibody Ab2 3+ dispersion, which is characterized by the following preparation steps:

[0057] Dissolve 45 mg of dopamine hydrochloride and 6.0 mg of ferric chloride hexahydrate in 130 mL of ultrapure water. After stirring for 1 h, quickly add 20 mL of aqueous solution containing 300 - 600 mg of tris(hydroxymethyl)aminomethane, and continue stirring for 2 h; perform centrifugal separation, wash the precipitate three times with ultrapure water to obtain SMNP-Fe 3+ nano-microspheres, and disperse them in 1.0 mL of ultrapure water for later use; take 1.0 mL of 18 mg / mL SMNP-Fe 3+ and 5 mL of 10 μg / mL Ab2 and add them to a centrifuge tube. After thoroughly mixing the solution, incubate it with shaking at 4°C for 10 h. Then, centrifuge and wash it, and disperse it evenly in 5 mL of PBS buffer solution with pH = 7.4 to obtain SMNP-Fe 3+ -Ab2 dispersion, which is stored in a refrigerator at 4°C for later use.

[0058] Example 6 Detection of SMNP-Fe labeled with antibody Ab2 3+ dispersion, which is characterized by the following preparation steps:

[0059] Dissolve 60 mg of dopamine hydrochloride and 8.0 mg of ferric chloride hexahydrate in 130 mL of ultrapure water. After stirring for 1 h, quickly add 20 mL of aqueous solution containing 600 mg of tris(hydroxymethyl)aminomethane, and continue stirring for 3 h; perform centrifugal separation, wash the precipitate three times with ultrapure water to obtain SMNP-Fe 3+ nano-microspheres, and disperse them in 1.0 mL of ultrapure water for later use; take 2.0 mL of 18 mg / mL SMNP-Fe 3+ and 10 mL of 10 μg / mL Ab2 and add them to a centrifuge tube. After thoroughly mixing the solution, incubate it with shaking at 4°C for 12 h. Then, centrifuge and wash it, and disperse it evenly in 10 mL of PBS buffer solution with pH = 7.4 to obtain SMNP-Fe 3+ -Ab2 dispersion, which is stored in a refrigerator at 4°C for later use.

[0060] Example 7 MgFe2O4 dispersion, which is characterized by the following preparation steps:

[0061] Mix 40 mL of ethylene glycol solution containing 0.1 g of polyvinylpyrrolidone with 1 mL of ethylene glycol solution containing 0.81 g of Fe(NO3)2·6H2O and 0.64 g of Mg(NO3)2·6H2O and stir well. Then add 0.1 g of polyethylene glycol and 1.0 g of sodium acetate. After ultrasonic mixing for 1 h, transfer the above solution to a high-pressure reactor and react at 200 °C for 24 h. After cooling to room temperature, wash with ethanol and ultrapure water by magnetic separation, and then dry in a vacuum drying oven for 12 h. Ultrasonically disperse the ground magnesium ferrite nanomaterial in ultrapure water to prepare a MgFe2O4 dispersion for standby.

[0062] Example 8 MgFe2O4 dispersion, characterized in that the preparation steps are as follows:

[0063] Mix 60 mL of ethylene glycol solution containing 0.1 g of polyvinylpyrrolidone with 10 mL of ethylene glycol solution containing 0.81 g of Fe(NO3)2·6H2O and 0.64 g of Mg(NO3)2·6H2O and stir well. Then add 1.0 g of polyethylene glycol and 3.0 g of sodium acetate. After ultrasonic mixing for 1 h, transfer the above solution to a high-pressure reactor and react at 200 °C for 24 h. After cooling to room temperature, wash with ethanol and ultrapure water by magnetic separation, and then dry in a vacuum drying oven for 12 h. Ultrasonically disperse the ground magnesium ferrite nanomaterial in ultrapure water to prepare a MgFe2O4 dispersion for standby.

[0064] Example 9 MgFe2O4 dispersion, characterized in that the preparation steps are as follows:

[0065] Mix 80 mL of ethylene glycol solution containing 0.1 g of polyvinylpyrrolidone with 20 mL of ethylene glycol solution containing 0.81 g of Fe(NO3)2·6H2O and 0.64 g of Mg(NO3)2·6H2O and stir well. Then add 2.0 g of polyethylene glycol and 6.0 g of sodium acetate. After ultrasonic mixing for 1 h, transfer the above solution to a high-pressure reactor and react at 200 °C for 24 h. After cooling to room temperature, wash with ethanol and ultrapure water by magnetic separation, and then dry in a vacuum drying oven for 12 h. Ultrasonically disperse the ground magnesium ferrite nanomaterial in ultrapure water to prepare a MgFe2O4 dispersion for standby.

[0066] Example 10 Detection of the concentration of lung cancer biomarker CYFRA21-1, characterized in that the operation steps are as follows: (1) Use an electrochemical workstation, with a saturated calomel electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared sensor as the working electrode to form a three-electrode system, and perform tests in a PBS bottom solution containing 0.15 mol / L ascorbic acid at pH = 7.4.

[0067] (2) Detection is carried out by the time - current method. The light source is full - wavelength, and the bias voltage is set to 0V;

[0068] (3) After the electrodes are placed, the light is turned on every 20s and irradiated continuously for 20s, and the photocurrent intensity is detected;

[0069] (4) Different concentrations of CYFRA21 - 1 standard solutions are used for determination. Record the changes in the photocurrent values corresponding to different concentrations of CYFRA21 - 1 standard solutions and plot the working curve;

[0070] (5) Test the photoelectrochemical sensor incubated with the actual sample of CYFRA21 - 1 with unknown concentration to obtain the corresponding signal intensity. Based on this, the concentration of CYFRA21 - 1 in the reagent sample can be calculated according to the working curve.

Claims

1. A preparation method of a multiple suppression quenching type photoelectrochemical sensor, characterized in that, It includes the following steps: (1) Ultrasonically clean the indium tin oxide (ITO) conductive glass electrode cut into a size of 2.0 cm × 0.7 cm successively in acetone, ultrapure water, absolute ethanol and ultrapure water for 1 h, and dry it with nitrogen. (2) Take 6 μL of a MgFe2O4 dispersion with a concentration of 2.0 - 6.0 mg / mL and drop it onto the conductive surface of the ITO electrode, and let it dry at room temperature. (3) Then dip the ITO electrode successively in 0.8 mol / L Cd(NO3)2 and 0.1 mol / L Na2S solutions for 30 s, repeat 2 - 10 times, and after drying at room temperature, CdS is formed and fully combined on the surface of MgFe2O4 to form MgFe2O4@CdS as the substrate material. (4) Then immerse the ITO electrode in a 1 - 5 mmol / L mercaptoacetic acid solution for 30 min to introduce carboxyl groups on the surface of MgFe2O4@CdS, and rinse it with ultrapure water. (5) Drop 3 μL of a 1:1 mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide with a concentration of 0.01 mol / L onto the surface of the above ITO electrode, and activate it at room temperature for 1 h; then continue to drop-coat 6 μL of a capture antibody Ab1 solution with a concentration of 10 μg / mL, incubate at room temperature for 1 h, and then rinse with a phosphate buffer solution (PBS) with pH = 7.

4. (6) Drop 3 μL of a bovine serum albumin solution (BSA) with a mass fraction of 0.01 - 1% to block the non-specific active sites on the electrode surface, incubate at room temperature for 30 min, and then rinse with a PBS solution with pH = 7.

4. (7) Then continue to drop 6 μL of a series of CYFRA21-1 solutions with different concentrations from 10 pg / mL to 100 ng / mL, rinse the electrode surface with ultrapure water, incubate at 37 °C for 0.5 - 2 h, and then rinse the electrode surface with a PBS with pH = 7.

4. (8) Add 6 μL of the dispersion of SMNP-Fe labeled with the detection antibody Ab2 with a concentration of 2.0 - 4.0 mg / mL, and rinse the surface of the electrode with PBS at pH = 7.4 to prepare a multiplex inhibition quenching type photoelectrochemical sensor. 3+ ​ 2. The preparation method of a multiple inhibition quenching type photoelectrochemical sensor as described in claim 1, the preparation of the dispersion liquid of the detection antibody Ab2-labeled SMNP-Fe 3+ , which is characterized in that The steps are as follows: Dissolve 30 - 60 mg of dopamine hydrochloride and 4.0 - 8.0 mg of ferric chloride hexahydrate in 130 mL of ultrapure water, stir for 1 h, then quickly add 20 mL of an aqueous solution containing 300 - 600 mg of tris(hydroxymethyl)aminomethane, and continue to stir for 1 - 3 h. Centrifugal separation was performed, and the precipitate was washed three times with ultrapure water to obtain SMNP-Fe 3+ nano-microspheres, which were dispersed in 1.0 mL of ultrapure water for later use; 0.5 - 2.0 mL of 18 mg / mL SMNP-Fe 3+ and 1 - 10 mL of 10 μg / mL Ab2 were added to a centrifuge tube. After the solution was thoroughly mixed, it was incubated with shaking at 4°C for 8 - 12 h, centrifuged and washed, and dispersed evenly in 0.5 - 10 mL of PBS buffer solution with pH = 7.4 to obtain SMNP-Fe 3+ -Ab2 dispersion, which was stored in a refrigerator at 4°C for later use.

3. The preparation method of a multiple inhibition quenching type photoelectrochemical sensor as described in claim 1, for the preparation of the MgFe2O4 dispersion liquid, is characterized in that, The steps are as follows: Mix 40 - 80 mL of an ethylene glycol solution containing 0.1 g of polyvinylpyrrolidone with 1 - 20 mL of an ethylene glycol solution containing 0.81 g of Fe(NO3)2·6H2O and 0.64 g of Mg(NO3)2·6H2O and stir evenly, then add 0.1 - 2.0 g of polyethylene glycol and 1.0 - 6.0 g of sodium acetate, and ultrasonically mix for 1 h; transfer the above solution to a high-pressure reaction kettle and react at 200 °C for 24 h; after cooling to room temperature, wash it with ethanol and ultrapure water by magnetic separation, dry it in a vacuum drying oven for 12 h, and ultrasonically disperse the ground magnesium ferrite nanomaterial in ultrapure water to prepare a MgFe2O4 dispersion for standby.

4. A detection method for a sensor prepared by the preparation method as described in claim 1, characterized in that, For the detection of the concentration of the lung cancer biomarker CYFRA21-1, the steps are as follows: (1) An electrochemical workstation was used to form a three-electrode system with a saturated calomel electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared sensor as the working electrode. The test was carried out in a PBS base solution with a pH of 7.4 containing 0.15 mol / L ascorbic acid. (2) Detection was carried out by the time-current method. The light source was a full wavelength, and the bias voltage was set to 0 V. (3) After the electrode was placed, the light was turned on every 20 s and irradiated continuously for 20 s to detect the photocurrent intensity. (4) Different concentrations of CYFRA21-1 standard solutions were used for determination. The changes in the photocurrent values corresponding to different concentrations of CYFRA21-1 standard solutions were recorded, and a working curve was plotted. (5) The photoelectrochemical sensor incubated with an actual sample of CYFRA21-1 with an unknown concentration was tested to obtain the corresponding signal intensity. Based on this, the concentration of CYFRA21-1 in the sample solution to be measured could be calculated according to the working curve.