CN111351934B - Marker immunosensor for diagnosing early lung cancer tumor and preparation method and application thereof - Google Patents

Marker immunosensor for diagnosing early lung cancer tumor and preparation method and application thereof Download PDF

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CN111351934B
CN111351934B CN202010164228.XA CN202010164228A CN111351934B CN 111351934 B CN111351934 B CN 111351934B CN 202010164228 A CN202010164228 A CN 202010164228A CN 111351934 B CN111351934 B CN 111351934B
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lung cancer
cancer tumor
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刘宁
吕静
郭婧
魏雪
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Affiliated Hospital of University of Qingdao
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Abstract

The invention discloses a marker immunosensor for diagnosing early lung cancer tumor and a preparation method and application thereof, the manganese dioxide/hollow nano gold ball compound is prepared by taking a hollow nano gold ball prepared by taking an ionic liquid as a ligand as a matrix, potassium permanganate as a manganese source, hexadecyl trimethyl ammonium bromide and polyethylene glycol as surfactants by adopting a liquid-phase coprecipitation method, and the electrochemical immunosensor used for diagnosing the early lung cancer tumor marker is prepared by taking the electrode modifier as an electrode modifier, the electrode is modified by the hollow gold nanospheres and the nano petal-shaped manganese dioxide together so as to cooperatively improve the specific surface area of the electrode and increase the adsorption capacity of the antibody, therefore, the sensitivity and the selectivity of the electrochemical immunosensor are obviously improved, the rapid, high-sensitivity and high-selectivity detection of the sample is realized, and the electrochemical immunosensor has wide market development prospect. The lung cancer tumor marker immunosensor is simple to prepare and convenient to operate.

Description

Marker immunosensor for diagnosing early lung cancer tumor and preparation method and application thereof
Technical Field
The invention relates to the technical field of novel nanometer functional materials and biosensors, in particular to an immunosensor for an early lung cancer tumor marker and a preparation method and application thereof.
Background
Lung cancer is one of the most life-threatening malignancies in humans, and its incidence and mortality rate increase the fastest. It has been reported that lung cancer has increased significantly over the last fifty years, with lung cancer incidence and mortality accounting for the first in male and second in female patients with malignancies. Therefore, early diagnosis and early treatment of lung cancer are key points for improving the survival rate of lung cancer patients. The detection of the lung cancer tumor marker can reflect the biological characteristics of the lung cancer, and has important significance for treatment, auxiliary diagnosis and prognosis judgment.
At present, a plurality of clinical detection methods of lung cancer tumor markers exist, such as an enzyme immunoassay method, a radioimmunoassay and the like, but the detection methods have a plurality of defects, such as easy enzyme inactivation in the enzyme immunoassay method, serious radioactive pollution in the radioimmunoassay and the like.
The immunosensor is a biosensor developed by the identification function of antigen-antibody specific binding, and the electrochemical immunosensor is the most diverse immunosensor, the earliest research and a more mature branch. The method has the advantages of high sensitivity, high analysis speed, small volume, good selectivity, suitability for integration and the like, so the method has good application prospects in environmental protection, clinical diagnosis, drug analysis and food safety detection. However, the technical difficulty of the electrochemical immunosensor is how to improve the magnitude of the current and the detection sensitivity and selectivity thereof.
Therefore, it is urgently needed to provide an environment-friendly lung cancer tumor marker sensor with high sensitivity, strong specificity and low requirement on working conditions.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides an immunosensor for a marker for diagnosing early lung cancer tumor, and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a lung cancer tumor marker immunosensor for diagnosing early stage lung cancer, the lung cancer tumor marker immunosensor comprising: the electrode comprises a working electrode, a reference electrode and a counter electrode, wherein a substrate electrode of the working electrode is a glassy carbon electrode, the surface of the glassy carbon electrode is sequentially modified with a manganese dioxide/hollow nano gold ball compound, a lung cancer tumor marker antibody and Bovine Serum Albumin (BSA), the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode.
The invention also provides a preparation method of the immunosensor for diagnosing the early lung cancer tumor marker, which comprises the following steps:
s1, preparing a manganese dioxide/hollow nano gold ball compound:
ultrasonically dispersing hollow nano gold spheres in aqueous solution of polyethylene glycol and hexadecyl trimethyl ammonium bromideAdding KMnO4The solution is naturally cooled to room temperature after the reaction is finished by adopting a liquid-phase coprecipitation method, and then the manganese dioxide/hollow nano gold ball compound is prepared after centrifugation, washing and drying;
s2, preparing the lung cancer tumor marker immunosensor working electrode.
To further limit the above, in step S1, KMnO4The dosage ratio of the hollow nano gold ball to the hollow nano gold ball is 1mmol (10-50) mg/mL.
To further limit the above, in step S1, KMnO4The molar use ratio of the ammonium bromide to the hexadecyl trimethyl ammonium bromide is 1: 2-5.
In a further limitation of the above aspect, in step S1, the mass concentration of the polyethylene glycol is 5 to 10%.
As a further limitation of the scheme, in the step S1, the reaction temperature of the liquid-phase coprecipitation method is 80-100 ℃ and the reaction lasts for 2-4 h.
As a further limitation of the above scheme, in step S1, the method for preparing the hollow nanogold ball is as follows:
a) preparation of 1, 3-bis (3-bromopropyl) imidazolium bromide
Adding 0.705g of sodium hydride into 30mL of acetonitrile solution containing 1.0g of imidazole in small amount for multiple times, reacting for three hours in an ice bath to form white acetonitrile suspension of imidazole sodium, dropwise adding the white acetonitrile suspension into the acetonitrile solution containing 11.876g of 1, 3-dibromopropane, stirring overnight at 55 ℃, removing acetonitrile by rotary evaporation after the reaction is finished, adding toluene for washing, and performing spin drying to obtain 1, 3-bis (3-bromopropyl) imidazole bromide;
b) 5mL of 30mmol/L aqueous 1, 3-bis (3-bromopropyl) imidazolium bromide solution was added to 5mL of 10mmol/L HAuCl with rapid stirring4In the process, 5mL of 60mmol/L newly prepared NaBH is quickly injected4And continuously stirring for reaction for 5min, centrifuging and washing for 3 times at 8000r/min, and storing at 4 ℃ for later use.
As a further limitation of the above scheme, in step S2, the specific steps of preparing the working electrode of the lung cancer tumor marker immunosensor are as follows:
s21, preprocessing the working electrode to make the surface smooth;
s22, 5-8 mu L of 2-8 mg/mL manganese dioxide/hollow nano gold ball compound is dripped on the surface of the electrode obtained in the step S21, and after the compound is dried to form a film, the film is cleaned by ultrapure water;
s23, dripping 5-8 mu L of lung cancer tumor marker antibody solution with the concentration of 10 mu g/mL on the surface of the electrode obtained in the step S22, storing and airing in a refrigerator at 4 ℃, cleaning by using a phosphate buffer solution, airing to form a film, and storing at 4 ℃;
s24, dripping 3-6 mu L of 1% BSA solution on the surface of the electrode prepared in the step S23 for sealing nonspecific active sites on the electrode, storing and airing in a refrigerator at 4 ℃, cleaning with phosphate buffer solution, airing and forming a film, and thus obtaining the working electrode of the lung cancer tumor marker immunosensor.
The invention also provides a lung cancer tumor marker immunosensor for diagnosing early lung cancer tumor markers, which is used for detecting the lung cancer tumor markers, and the method comprises the following steps:
1) adding a known-concentration lung cancer tumor marker antigen standard solution into a PBS (phosphate buffer solution) buffer solution of 40-60 mu L, pH-7.4 to prepare an antigen mixed solution, dropwise coating 5-10 mu L of the antigen mixed solution on a working electrode of the prepared lung cancer tumor marker immunosensor, storing and airing in a refrigerator at 4 ℃, cleaning the PBS buffer solution with the pH of 7.4, airing, and storing at 4 ℃;
2) a calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the working electrode assembled in the step 1) form a three-electrode system which is connected to an electrochemical workstation at the concentration of 5.0mmol/L [ Fe (CN))6]3-/[Fe(CN)6]4-+0.1mol/L KCl; detecting a current response difference value before and after the assembled working electrode recognizes the antigen through a differential pulse voltammetry; drawing a working curve according to the relation between the obtained current response difference and the concentration of the tumor marker antigen standard solution;
3) and replacing the standard solution of the lung cancer tumor marker antigen with the sample solution to be detected, and detecting according to the drawing method of the working curve of the lung cancer tumor marker antigen.
As a further definition of the above aspect, the tumor marker of lung cancer is selected from one of the following: carcinoembryonic antigen (CEA), carbohydrate antigen (CA-125), carbohydrate antigen (CA-242), neuron-specific enolase (NSE), Tissue Polypeptide Antigen (TPA), Squamous Cell Carcinoma Antigen (SCCA).
As a further definition of the above, the tumor marker of lung cancer is selected from Squamous Cell Carcinoma Antigen (SCCA).
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention takes the hollow nano gold ball prepared by taking the ionic liquid as the ligand as the matrix and potassium permanganate (KMnO)4) The manganese dioxide/hollow nano gold sphere compound is prepared by a liquid-phase coprecipitation method as a manganese source, cetyl trimethyl ammonium bromide and polyethylene glycol are used as surfactants, the manganese dioxide/hollow nano gold sphere compound is used as an electrode modifier to prepare the electrochemical immunosensor which is low in cost, high in sensitivity, good in specificity and capable of rapidly detecting early lung cancer tumor markers.
(2) The lung cancer tumor marker immunosensor provided by the invention is simple to prepare, convenient to operate, low in detection environment requirement, capable of realizing rapid, sensitive and high-selectivity detection of a sample, and wide in market development prospect.
Drawings
Fig. 1 is a scanning electron microscope characterization of the manganese dioxide/hollow nanogold ball composite prepared in example 1.
FIG. 2 is a diagram of the results of Electrochemical Impedance Spectroscopy (EIS) characterization of the modified electrode obtained in each step of example 1.
FIG. 3 is a graph of the linear relationship of the electrochemical immunosensor prepared in example 1 for various concentrations of squamous cell carcinoma antigen.
Fig. 4 is a graph showing the experimental results of the anti-interference performance of the electrochemical immunosensor prepared in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly apparent, the present invention is further described in detail with reference to the following embodiments; it should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention; reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The present invention will be described in further detail below with reference to specific embodiments and with reference to the attached drawings.
Example 1
A lung cancer tumor marker immunosensor for diagnosing early stage lung cancer, the lung cancer tumor marker immunosensor comprising: the device comprises a working electrode, a reference electrode and a counter electrode, wherein a substrate electrode of the working electrode is a glassy carbon electrode, the surface of the glassy carbon electrode is sequentially modified with a manganese dioxide/hollow nano gold ball compound, a squamous cell carcinoma antibody and Bovine Serum Albumin (BSA), the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode.
The preparation method of the immunosensor for diagnosing the early lung cancer tumor marker comprises the following steps:
s1, preparing a manganese dioxide/hollow nano gold ball compound:
ultrasonically dispersing 500mg of hollow nano gold spheres in 30mL of aqueous solution of polyethylene glycol with the mass concentration of 8% and 0.1mol/L hexadecyl trimethyl ammonium bromide, and adding 0.158g of KMnO4Reacting the solution at 90 ℃ for 3 hours by adopting a liquid-phase coprecipitation method, naturally cooling to room temperature after the reaction is finished, and then centrifuging, washing and drying to obtain a manganese dioxide/hollow nanogold ball compound;
s2, preparing a lung cancer tumor marker immunosensor working electrode:
s21, preprocessing the working electrode to make the surface smooth;
s22, dripping 6 mu L of 5mg/mL manganese dioxide/hollow nano gold ball compound aqueous dispersion on the surface of the electrode obtained in the step S21, airing to form a film, and then cleaning with ultrapure water;
s23, dripping 6 mu L of squamous cell carcinoma antibody solution with the concentration of 10 mu g/mL on the surface of the electrode obtained in the step S22, storing and airing in a refrigerator at 4 ℃, cleaning by using a phosphate buffer solution, airing to form a film, and storing at 4 ℃;
s24, dripping 6 mu L of 1% BSA solution on the surface of the electrode prepared in the step S23 to seal the nonspecific active sites on the electrode, storing and airing in a refrigerator at 4 ℃, cleaning with phosphate buffer solution, airing to form a film, and obtaining the working electrode of the lung cancer tumor marker immunosensor;
s3, adding a known concentration of lung cancer tumor marker antigen standard solution into PBS buffer solution with the concentration of 50 mu L, pH-7.4 to prepare a squamous cell carcinoma antigen mixed solution with the concentration of 0.01, 0.1, 0.5, 1, 2.5, 5, 8, 10, 12ng/mL in sequence, dripping 6 mu L of the squamous cell carcinoma antigen mixed solution on a working electrode of the prepared lung cancer tumor marker immunosensor, storing and airing the working electrode in a refrigerator at 4 ℃, cleaning and airing the working electrode in the PBS buffer solution with the pH of 7.4, and storing the working electrode at 4 ℃;
s4, forming a three-electrode system by a calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the working electrode assembled in the step 1), connecting the three-electrode system to an electrochemical workstation at the concentration of 5.0mmol/L [ Fe (CN))6]3-/[Fe(CN)6]4-+0.1mol/L KCl; detecting a current response difference value before and after the assembled working electrode recognizes the antigen through a differential pulse voltammetry; and drawing a working curve according to the relation between the obtained current response difference and the concentration of the tumor marker antigen standard solution.
The preparation method of the hollow nano gold ball comprises the following steps:
a) preparation of 1, 3-bis (3-bromopropyl) imidazolium bromide
Adding 0.705g of sodium hydride into 30mL of acetonitrile solution containing 1.0g of imidazole in small amount for multiple times, reacting for three hours in an ice bath to form white acetonitrile suspension of imidazole sodium, dropwise adding the white acetonitrile suspension into the acetonitrile solution containing 11.876g of 1, 3-dibromopropane, stirring overnight at 55 ℃, removing acetonitrile by rotary evaporation after the reaction is finished, adding toluene for washing, and performing spin drying to obtain 1, 3-bis (3-bromopropyl) imidazole bromide;
b) 5mL of 30 mmol/L1, 3-bis (3-bromopropyl) imidazole bromide aqueous solution is added under the condition of rapid stirring5mL of 10mmol/L HAuCl was added4In the process, 5mL of 60mmol/L newly prepared NaBH is quickly injected4And continuously stirring for reaction for 5min, centrifuging and washing for 3 times at 8000r/min, and storing at 4 ℃ for later use.
Fig. 1 is a scanning electron microscope characterization result of the manganese dioxide/hollow nanogold ball composite prepared in this example, and it can be seen from the figure that the hollow nanogold balls are fused with flower-like structures formed by stacking manganese dioxide lamellar structures, which illustrates that the hollow nanogold balls are successfully composited with manganese dioxide.
As shown in fig. 2, the modified electrode obtained in each step of this example was characterized by electrochemical alternating current impedance spectroscopy (EIS), which is one of the effective tools for exploring the properties of chemically modified electrode interface. The spectrogram is generally divided into a high-frequency part and a low-frequency part, wherein the high-frequency part is a dynamic control area, and the low-frequency part is a diffusion control area. At 5.0 mmol. multidot.L-1K3[Fe(CN)6]/K4[Fe(CN)6](1:1)+0.1mol·L-1PBS(pH＝7.0)+0.1mol·L-1Alternating current impedance characterization is carried out in a KCl solution, and charge transfer resistance of an electrode interface is calculated by using a Randlescirt fitting circuit, wherein the interface charge transfer resistance (Rct) and a diffusion resistance (ZW) are connected with an interface capacitance (Cdl) in parallel, and the diameter of a semicircle corresponds to the interface charge transfer resistance (Rct). The Nyquist curve is shown in fig. 2, and it can be seen that the ac impedance spectrum of the bare electrode (curve a) is very small in the semicircle of the high frequency part, the clean glassy carbon electrode (a) is the smallest in the semicircle, the charge transfer resistance on the surface of the electrode is increased after the manganese dioxide/hollow nanogold complex is modified (b), the squamous cell carcinoma antibody is fixed (c), the diameter of the semicircle is increased because the biomacromolecule is not favorable for the conduction of electrons, which can prevent the transfer of electrons between the electrode interface and the solution, the impedance semicircle diameter is continuously increased after the non-specific binding site on the electrode surface is blocked by BSA (d), because the excess aldehyde group is combined with the amino group on the BSA, and the impedance semicircle diameter is further increased after the electrochemical immunosensor is specifically combined with the squamous cell carcinoma antigen (e). The results show that the electrochemical immunosensor for squamous cell carcinoma is successfully prepared after gradual modification。
FIG. 3 is a linear relationship curve of the electrochemical immunosensor prepared in this example for different concentrations of squamous cell carcinoma antigens. As can be seen from the graph, when the concentration of the squamous cell carcinoma antigen is in the range of 0.01-10 ng/mL, the corresponding difference value delta I of the currents before and after the antigen is identified by the working electrode and the concentration C (ng/mL) of the squamous cell carcinoma antigen have a good linear relation, and the linear equation is that the delta I is 2.5147C +36.982, R2The detection limit was 10.3pg/mL (S/N was 3) at 0.999. The result shows that the electrochemical immunosensor constructed on the basis of the manganese dioxide/hollow nano gold ball compound modified electrode can be used for high-sensitivity detection of squamous cell carcinoma antigens so as to be used for early clinical diagnosis of lung cancer.
The anti-interference performance is one of important indexes for measuring the practicability of the electrochemical sensor. In order to examine the specific recognition performance of the electrochemical immunosensor for squamous cell carcinoma prepared in the embodiment of the invention, four tumor markers of solutions of neuron-specific enolase (NSE), Albumin (ALB), lysine (Lys) and glucose (Glu) are selected as interference items for selective experiments. 1ng/mL of squamous cell carcinoma was mixed with 50ng/mL of NSE, ALB, Lys, Glu solutions, respectively, and electrochemical performance test was performed under the optimal conditions, and the results are shown in FIG. 4. As can be seen from the figure: the current response difference of the electrochemical immunosensor before and after the interferent is added has no obvious change, so that the electrochemical immunosensor prepared by the method has good selectivity.
In order to further examine the repeatability of the squamous cell carcinoma electrochemical immunosensor prepared by the invention, five working electrodes are simultaneously prepared and used for testing the current change condition of a squamous cell carcinoma antigen solution with the concentration of 1 ng/mL. The relative standard deviation of the measurement results of the five sensors is calculated to be 3.4%, which shows that the electrochemical immunosensor prepared by the invention has good repeatability.
To investigate the stability of the prepared immunosensor. We placed the prepared sensor in phosphate buffer solution with pH 7.4, stored in a refrigerator at 4 ℃ for use, and used to test the change in current of a squamous cell carcinoma antigen solution with a concentration of 1ng/mL after 2 weeks. Experiments show that the current response difference is 95.2% of the initial value, which indicates that the electrochemical immunosensor has better stability and can better maintain the biological activity of the antibody.
Examples 2 to 6
A preparation method of a lung cancer tumor marker immunosensor comprises the following steps: five lung cancer tumor markers including CEA, CA-242, CA-125, NSE or TPA are selected, the lung cancer tumor marker immunosensor is constructed and detected according to the steps described in the embodiment 1, and the detection technical indexes of the lung cancer tumor markers are shown in Table 1.
TABLE 1 detection technical index of lung cancer tumor marker
Tumor marker Linear range (ng/mL) Detection Limit ((pg/mL))
CEA 0.01～10 2.1
CA-242 0.01～20 10.2
CA-125 0.01～10 12.5
NSE 0.01～20 3.8
TPA 0.01～20 2.8
Example 7
The present embodiment provides a method for detecting a tumor marker of lung cancer in a human serum sample by using the electrochemical immunosensor prepared in embodiment 1, the method comprising:
accurately transferring a human serum sample, adding a lung cancer tumor marker antigen standard solution with a certain mass concentration, taking human serum without the lung cancer tumor marker antigen as a blank, carrying out a labeling recovery experiment, detecting according to the steps of example 1, and determining the recovery rate of the lung cancer tumor marker in the sample, wherein the detection result is shown in table 2.
TABLE 2 detection results of lung cancer tumor markers in human serum
Figure GDA0002452444640000091
As can be seen from the detection results in Table 2, the Relative Standard Deviation (RSD) of the results is less than 2.5%, and the average recovery rate is 98.8-102.2%, which indicates that the method provided by the invention can be used for detecting lung cancer tumor markers in human serum, and the method provided by the invention has the advantages of high sensitivity, strong specificity, and accurate and reliable results.
While the invention has been described with respect to specific embodiments thereof, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention; those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and alterations of the above embodiments according to the spirit and techniques of the present invention are also within the scope of the present invention.

Claims (2)

1. A lung cancer tumor marker immunosensor for diagnosing an early stage lung cancer tumor, comprising: the electrode comprises a working electrode, a reference electrode and a counter electrode, wherein a substrate electrode of the working electrode is a glassy carbon electrode, the surface of the glassy carbon electrode is sequentially modified with a manganese dioxide/hollow nano gold ball compound, a lung cancer tumor marker antibody and Bovine Serum Albumin (BSA), the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum wire electrode;
the preparation method of the sensor comprises the following steps:
s1, preparing a manganese dioxide/hollow nano gold ball compound:
ultrasonically dispersing hollow nano gold spheres in aqueous solution of polyethylene glycol and hexadecyl trimethyl ammonium bromide, and adding KMnO4The solution is naturally cooled to room temperature after the reaction is finished by adopting a liquid-phase coprecipitation method, and then the manganese dioxide/hollow nano gold ball compound is prepared after centrifugation, washing and drying;
s2, preparing a lung cancer tumor marker immunosensor working electrode;
in step S1, KMnO4The dosage ratio of the hollow nano gold ball to the hollow nano gold ball is 1mmol (10-50) mg/mL;
in step S1, KMnO4The molar use ratio of the ammonium bromide to the hexadecyl trimethyl ammonium bromide is 1: 2-5;
in the step S1, the mass concentration of the polyethylene glycol is 5-10%;
in the step S1, the reaction temperature of the liquid-phase coprecipitation method is 80-100 ℃ and the reaction lasts for 2-4 h;
in step S2, the specific steps of preparing the working electrode of the lung cancer tumor marker immunosensor are as follows:
s21, preprocessing the working electrode to make the surface smooth;
s22, 5-8 mu L of 2-8 mg/mL manganese dioxide/hollow nano gold ball compound is dripped on the surface of the electrode obtained in the step S21, and after the compound is dried to form a film, the film is cleaned by ultrapure water;
s23, dripping 5-8 mu L of lung cancer tumor marker antibody solution with the concentration of 10 mu g/mL on the surface of the electrode obtained in the step S22, storing and airing in a refrigerator at 4 ℃, cleaning by using a phosphate buffer solution, airing to form a film, and storing at 4 ℃;
s24, dripping 3-6 mu L of 1% BSA solution on the surface of the electrode prepared in the step S23 for sealing the nonspecific active sites on the electrode, storing and airing in a refrigerator at 4 ℃, cleaning with phosphate buffer solution, airing and forming a film, and thus obtaining the working electrode of the lung cancer tumor marker immunosensor.
2. The immunosensor for diagnosing tumor markers of early stage lung cancer according to claim 1, wherein the immunosensor is used for detecting the tumor markers of the lung cancer by the following method:
1) adding a known concentration of a lung cancer tumor marker antigen standard solution into a PBS buffer solution with the concentration of 40-60 mu L, pH =7.4 to prepare an antigen mixed solution, dropwise coating 5-10 mu L of the antigen mixed solution on a working electrode of the prepared lung cancer tumor marker immunosensor, storing and airing in a refrigerator at 4 ℃, cleaning and airing the PBS buffer solution with the pH =7.4, and storing at 4 ℃;
2) a calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the working electrode assembled in the step 1) form a three-electrode system which is connected to an electrochemical workstation at the concentration of 5.0mmol/L [ Fe (CN))6]3-/[Fe(CN)6]4-+0.1mol/L KCl; detecting a current response difference value before and after the assembled working electrode recognizes the antigen through a differential pulse voltammetry; drawing a working curve according to the relation between the obtained current response difference and the concentration of the tumor marker antigen standard solution;
3) and replacing the standard solution of the lung cancer tumor marker antigen with the sample solution to be detected, and detecting according to the drawing method of the working curve of the lung cancer tumor marker antigen.
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