Double-channel gold-copper-doped graphene electrochemical immunosensor as well as preparation method and application thereof

Abstract

The invention discloses a double-channel gold-copper-doped graphene electrochemical immunosensor as well as a preparation method and application thereof. The double-channel gold-copper-doped graphene electrochemical immunosensor comprises a gold / gold-copper co-doped vertical graphene electrode, and bovine serum albumin and an antibody which are incubated on the gold / gold-copper co-doped vertical graphene electrode, the gold / gold-copper co-doped vertical graphene electrode comprises a gold-copper co-doped vertical graphene electrode and gold nanoparticles loaded on the surface of the gold-copper co-doped vertical graphene electrode, and the gold-copper co-doped vertical graphene electrode comprises a substrate and a gold-copper co-doped vertical graphene nanosheet grown on the surface of the substrate. The sensor capable of sensitively detecting the concentration of the carcinoembryonic antigen secreted by the tumor cells is prepared, an immunoreaction detection system is built, a reliable industrial signal amplification platform is provided for early diagnosis of tumors, and a new strategy is developed for research of tumor cytology.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical sensors, and in particular relates to a dual-channel gold-copper doped graphene electrochemical immunosensor and a preparation method and application thereof. Background Art

[0002] Cancer is the second most life-threatening disease, and the survival rate of patients is very low. Tumor markers have become routine clinical examination items for auxiliary diagnosis, efficacy evaluation, and recurrence monitoring of malignant tumors. Therefore, rapid detection of carcinoembryonic antigen (CEA) in human tissues and body fluids is of great significance for early diagnosis, efficacy evaluation, and prognosis tracking of cancer. At present, diagnostic imaging methods are the most common means for identifying tumor patients. In addition, common tumor marker detection methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, dual-mode photothermal / chemiluminescence, surface-enhanced Raman spectroscopy, radioimmunology, and electrochemical immunoassay. Electrochemical immunoassay can use electrochemical immunosensors, which are a new type of biosensor based on antigen-antibody specific reactions. Due to its high sensitivity, low cost, and rapid response, it has attracted much attention in the field of clinical tumor diagnosis. However, traditional electrochemical immunosensors have limitations, complex compositions, and are difficult to achieve industrial mass production, resulting in low stability of electrochemical immunosensors. Summary of the invention

[0003] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preparing a gold-copper co-doped vertical graphene electrode, wherein the preparation method realizes the in-situ growth of gold-copper co-doped vertical graphene by electron-assisted hot-filament chemical vapor deposition (EA-HF-CVD).

[0004] Another object of the present invention is to provide a gold / gold-copper co-doped vertical graphene electrode, in which the gold (Au) nanoparticles in the gold / gold-copper co-doped vertical graphene electrode and the gold nanoparticles and copper nanoparticles doped in the gold-copper co-doped vertical graphene electrode act together as nucleation sites, providing abundant binding sites for antibodies (Ab).

[0005] Another object of the present invention is to provide a dual-channel gold-copper doped graphene electrochemical immunosensor, which adopts a dual-channel detection method and utilizes an electrochemical redox strategy of probe ions to detect immune tumor markers.

[0006] The purpose of the present invention is achieved through the following technical solutions.

[0007] A gold / gold-copper co-doped vertical graphene electrode comprises: a gold-copper co-doped vertical graphene electrode and gold (Au) nanoparticles loaded on the surface of the gold-copper co-doped vertical graphene electrode. The gold-copper co-doped vertical graphene electrode comprises: a substrate and gold-copper co-doped vertical graphene nanosheets grown on the surface of the substrate.

[0008] In the above technical solution, the material of the substrate is titanium or tantalum.

[0009] A method for preparing a gold / gold-copper co-doped vertical graphene electrode comprises: using the gold-copper co-doped vertical graphene electrode as a working electrode in a three-electrode system, immersing the three-electrode system in a tetrachloroauric acid aqueous solution, and using a multi-potential step electrochemical deposition method to deposit Au in the tetrachloroauric acid. 3+ The gold nanoparticles are electrochemically reduced to gold nanoparticles and loaded on the surface of a gold-copper co-doped vertical graphene electrode to obtain a gold / gold-copper co-doped vertical graphene electrode. The gold-copper co-doped vertical graphene electrode comprises a substrate and gold-copper co-doped vertical graphene nanosheets grown on the surface of the substrate.

[0010] In the above technical solution, the concentration of tetrachloroauric acid in the tetrachloroauric acid aqueous solution is 0.8-1.2 mM.

[0011] In the above technical solution, the three-electrode system includes: a working electrode, a counter electrode and a reference electrode, the counter electrode (CE) is a platinum sheet, and the reference electrode (RE) is a saturated calomel electrode (SCE).

[0012] In the above technical solution, the multi-potential step electrochemical deposition method has 5 to 100 cycles of electrodeposition, and each cycle of electrodeposition includes: firstly at a potential of 1 to 2V for 1 to 2s, and then at a potential of -2 to -1V for 4 to 6s.

[0013] A method for preparing a gold-copper co-doped vertical graphene electrode comprises the following steps:

[0014] Step 1: immerse the substrate in the first solution, and use the photocatalytic method to catalyze Au in tetrachloroauric acid in the first solution on the surface of the substrate. 3+ In situ reduction to gold nanoparticles, Cu in copper sulfate 2+ In-situ reduction to copper nanoparticles, obtaining a gold-copper catalyst precursor layer on the surface of the substrate, the substrate and the gold-copper catalyst precursor layer on its surface are used as the first substance, wherein the first solution comprises: tetrachloroauric acid (HAuCl4), copper sulfate (CuSO4) and water, the ratio of gold in tetrachloroauric acid to copper in copper sulfate is 1: (0.8-1.2) in terms of the amount of substance, and the concentration of tetrachloroauric acid in the first solution is 0.05-0.15 mM;

[0015] In step 1, the photocatalytic method uses infrared lamps.

[0016] In step 1, the concentration of copper sulfate in the first solution is 0.05-0.15 mM.

[0017] In step 1, the substrate is first immersed in the first solution for 20 to 40 minutes, and then the photocatalytic method is performed.

[0018] In step 1, the substrate is first polished with fine sandpaper before use, then ultrasonicated in water and anhydrous ethanol in sequence, and finally dried.

[0019] In step 1, the photocatalytic method includes: using an infrared lamp with a power of 370-380W to irradiate the first solution and the substrate immersed therein for at least 20 minutes.

[0020] In step 1, the substrate has a length of 29 to 31 mm, a width of 1.5 to 1.8 mm, and a thickness of 0.15 to 0.25 mm.

[0021] Step 2, placing the first substance obtained in step 1 on the sample stage of the reaction chamber in the hot wire CVD and diamond combined deposition system, sealing the reaction chamber, evacuating the reaction chamber to below 5 Pa, and continuously introducing methane (CH4) and hydrogen (H2) so that the volume ratio of methane to hydrogen is (15-20): (250-350) (the flow rate of the introduced methane is 15-20 mL min -1 The flow rate of hydrogen is 250~350mL min -1 ), when the pressure in the reaction chamber reaches 1600-1700Pa, apply 110-130A AC to the filament in the reaction chamber, and when the pressure in the reaction chamber reaches 4500-5500Pa, continue to apply 110-130A AC for 30-40min at this pressure (4500-5500Pa), and then adjust the flow rate of methane (CH4) and hydrogen so that the volume ratio of methane (CH4) and hydrogen is (30-50): (15-25) (adjust the flow rate of methane (CH4) to 30-50mL min -1 , adjust the flow rate of hydrogen (H2) to 15~25mL min -1 ), when the pressure of the reaction chamber reaches 300-500 Pa, a DC bias of 5-8 A is applied between the sample stage and the filament for 3-10 min to form gold-copper co-doped vertical graphene nanosheets on the surface of the substrate to obtain gold-copper co-doped vertical graphene electrodes, wherein when the DC bias is applied, the DC bias and the AC power together keep the reaction chamber at 900-1100° C.

[0022] In step 2, the filament is a tantalum filament.

[0023] A dual-channel gold-copper doped graphene electrochemical immunosensor comprises: a gold / gold-copper co-doped vertical graphene electrode and bovine serum albumin (BSA) and an antibody (Ab) incubated on the gold / gold-copper co-doped vertical graphene electrode.

[0024] The method for preparing the dual-channel gold-copper doped graphene electrochemical immunosensor comprises the following steps:

[0025] S1, incubating the antibody on the gold / gold-copper co-doped vertical graphene electrode to obtain Ab@gold / gold-copper co-doped vertical graphene electrode;

[0026] S1 specifically comprises: immersing the gold / gold-copper co-doped vertical graphene electrode in an antibody (Ab) solution and incubating for 30 to 90 minutes to obtain an Ab@gold / gold-copper co-doped vertical graphene electrode, wherein the antibody (Ab) solution comprises: a phosphate buffer solution (PBS) and an antibody (Ab), and the concentration of the antibody (Ab) in the antibody (Ab) solution is 100 to 5000 ng·mL -1 ;

[0027] S2, incubation of bovine serum albumin (BSA) on Ab@Au / AuCu co-doped vertical graphene electrode;

[0028] S2 specifically includes: immersing the Ab@gold / gold-copper co-doped vertical graphene electrode in a bovine serum albumin (BSA) solution and incubating for 10 to 20 minutes to obtain a dual-channel gold-copper doped graphene electrochemical immunosensor, wherein the bovine serum albumin (BSA) solution includes: phosphate buffer (PBS) and bovine serum albumin.

[0029] In S2, the concentration of bovine serum albumin (BSA) in the bovine serum albumin (BSA) solution is 1.4-1.6 wt %.

[0030] In S1 and S2, the incubation temperature was 36-38°C.

[0031] In S1 and S2, the pH of the phosphate buffer solution (PBS) is 7.35-7.45.

[0032] In the above technical scheme, the Ab@gold / gold-copper co-doped vertical graphene electrode was used as a dual-channel gold-copper doped graphene electrochemical immunosensor within 7 days after incubating bovine serum albumin (BSA) on the Ab@gold / gold-copper co-doped vertical graphene electrode.

[0033] A method for detecting the concentration of an antigen, comprising:

[0034] 1) preparing N standard solutions, each of which is a mixture of an antigen and a phosphate buffer solution (PBS) (the antigen in the standard solution is the same as the antigen to be detected in the antigen concentration detection method of the present invention), and the antigen (Ag) concentrations in the N standard solutions are different, and each standard solution is used as a sample solution to calculate ΔI, wherein the method for calculating ΔI of each sample solution includes:

[0035] ① Incubating antigens on the dual-channel gold-copper-doped graphene electrochemical immunosensor with a sample solution to obtain a sensor; performing a differential pulse voltammetry (DPV) test on the sensor to obtain a current value corresponding to a peak value of the DPV curve as I2;

[0036] ② Calculate ΔI of the sample solution, ΔI = I1-I2, where I1 is the current value corresponding to the peak value of the DPV curve obtained by differential pulse voltammetry (DPV) test of the dual-channel gold-copper doped graphene electrochemical immunosensor;

[0037] The antibodies on the dual-channel gold-copper-doped graphene electrochemical immunosensors in ① and ② correspond to the antigens.

[0038] In ①, the incubation temperature of the antigen is 36-38°C, and the incubation time of the antigen is 10-40 minutes.

[0039] In ①, the differential pulse voltammetry (DPV) test is an oxidation DPV test or a reduction DPV test.

[0040] 2) Establishing a coordinate system, taking the lg value and ΔI of the antigen concentration as the horizontal and vertical coordinates, substituting the lg value and ΔI of the antigen (Ag) concentration of N portions of the standard solution into the coordinate system, and fitting to obtain a linear regression equation;

[0041] 3) The solution to be tested is used as the sample solution, the ΔI of the solution to be tested is calculated, and the ΔI of the solution to be tested is substituted into the linear regression equation to obtain the lg value of the antigen concentration in the solution to be tested, and then the antigen concentration in the solution to be tested is calculated.

[0042] In the above technical solution, when performing differential pulse voltammetry (DPV) testing, the corresponding dual-channel gold-copper-doped graphene electrochemical immunosensor / sensor is immersed in a probe solution;

[0043] In the above technical scheme, the probe solution includes: potassium chloride (KCl), potassium ferrocyanide (K3[Fe(CN)6]) and potassium ferrocyanide (K4[Fe(CN)6]), the concentration of KCl in the probe solution is 0.09~0.11M, and the concentrations of K3[Fe(CN)6] and K4[Fe(CN)6] in the probe solution are 4.9~5.1mM respectively.

[0044] A method for detecting tumor marker concentration, comprising: obtaining the antigen concentration in the test solution as c1 according to the antigen concentration detection method based on the oxidation DPV test, obtaining the antigen concentration in the test solution as c2 according to the antigen concentration detection method based on the reduction DPV test, and taking the value of 0.5(c1+c2) as the tumor marker concentration.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The present invention uses a photocatalytic method to form gold nanoparticles and copper nanoparticles on a substrate, and under the action of an electric field in chemical vapor deposition, gold-copper co-doped vertical graphene nanosheets are formed on the surface of the substrate;

[0047] (2) The method of the present invention for detecting tumor marker concentrations uses dual-channel synergistic analysis of oxidation DPV test and reduction DPV test, which has high reliability;

[0048] (3) Compared with the traditional sandwich-type electrochemical immunosensor, the method for preparing the dual-channel gold-copper-doped graphene electrochemical immunosensor of the present invention is simpler to operate;

[0049] (4) The dual-channel gold-copper doped graphene electrochemical immunosensor prepared by the present invention has a wide linear range (can be used for concentrations of 1fg mL -1 ~30ng mL -1 The advantages include high long-term stability and low detection limit.

[0050] (5) The dual-channel gold-copper doped graphene electrochemical immunosensor prepared in the present invention can be used to detect the concentration of antigens released by tumor cells.

[0051] The present invention adopts the method of electrochemical gold deposition to electro-deposit gold nanoparticles onto the gold-copper co-doped vertical graphene electrode to prepare a sensor that can sensitively detect the concentration of carcinoembryonic antigen secreted by tumor cells, build an immune response detection system, provide a reliable industrial signal amplification platform for early diagnosis of tumors, and open up new strategies for tumor cytology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Surface SEM of the gold-copper co-doped vertical graphene electrode prepared in Example 1;

[0053] Figure 2 This is a cross-sectional SEM of the gold-copper co-doped vertical graphene electrode prepared in Example 1;

[0054] Figure 3 is a cyclic voltammetry scan rate variation diagram in Example 2, Figure 3a~g are the cyclic voltammetry scan rate change diagrams of Comparative Example 6, Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 1, Comparative Example 4 and Comparative Example 5, respectively;

[0055] Figure 4 is the Nyquist curve in Example 2, Figure 4 a~g are Nyquist curves of Comparative Example 6, Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 1, Comparative Example 4 and Comparative Example 5, respectively;

[0056] Figure 5 is the electrochemical correlation constant in Example 2, wherein, Figure 5 a is the liquid-solid interface charge transfer resistance of Example 1 and Comparative Examples 1 to 6, Figure 5 b is the effective electroactive surface area of ​​Example 1 and Comparative Examples 1 to 6, Figure 5 c is the electron transfer rate of Example 1 and Comparative Examples 1 to 6;

[0057] Figure 6 Surface SEM of the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3;

[0058] Figure 7 This is a cross-sectional SEM of the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3;

[0059] Figure 8 The Raman spectrum of the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3;

[0060] Fig. 9 X-ray photoelectron spectroscopy (XPS) of the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3;

[0061] Fig.10 a is the oxidation channel DPV curve of the gold-copper co-doped vertical graphene electrode as the working electrode in Example 4 and the oxidation channel DPV curve of the Ab@gold-copper co-doped vertical graphene electrode as the working electrode in Example 5, Fig.10 b is the oxidation channel DPV curve of the gold / gold-copper co-doped vertical graphene electrode as the working electrode in Example 4 and the oxidation channel DPV curve of the Ab@gold / gold-copper co-doped vertical graphene electrode as the working electrode in Example 5;

[0062] Fig.11 is the DPV curve in Example 7;

[0063] Fig.12 are the linear regression equations for the oxidation channel and the reduction channel;

[0064] Fig.133D images of the gold-copper co-doped vertical graphene electrode in Example 1, the gold / gold-copper co-doped vertical graphene electrode in Example 3, the Ab@gold / gold-copper co-doped vertical graphene electrode in Example 5, the dual-channel gold-copper doped graphene electrochemical immunosensor in Example 6, and the sensor in Example 7 under an atomic force microscope;

[0065] Fig.14 is the DPV curve in Example 8;

[0066] Fig.15 c in Example 8 Ox 、c Re With c ELISA comparison. DETAILED DESCRIPTION

[0067] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0068] The names, specifications and manufacturer information of the reagents / materials involved in the following examples are as follows:

[0069]

[0070]

[0071] The instruments and their model information involved in the following embodiments are as follows:

[0072] Instrument Name Instrument Manufacturer Electronic balance Mettler Toledo Instruments (Shanghai) Co., Ltd. CHI 660E Electrochemical Workstation Shanghai Chenhua Instrument Co., Ltd., China Saturated calomel electrode Tianjin Lanlike Chemical High-Tech Co., Ltd. Ultrasonic cleaning machine Shenzhen Jiemeng Cleaning Equipment Co., Ltd. Hot Filament CVD and Diamond Combined Deposition System Chinese Academy of Sciences Shenyang Scientific Instrument Co., Ltd. JDCY-20-500 AC filament power supply Chinese Academy of Sciences Shenyang Scientific Instrument Co., Ltd. DL-18 closed circuit cooling water unit Donggang Cooling Water Equipment Factory Cell culture incubator Thermo Fisher Scientific Roller machine SG Shandong Ruina Chuang Machinery Equipment Co., Ltd.

[0073] In the following examples, the cell culture medium consists of 89 wt % RPMI 1640 medium (from Thermo Fisher Scientific), 1 wt % penicillin-streptomycin mixture (from Beijing Solebow Technology Co., Ltd., Cat. No. P1400, containing 10000 U mL penicillin). -1 , containing streptomycin 10000 μg mL -1 ) and 10 wt % fetal bovine serum (from Beijing Solebow Technology Co., Ltd., S9020).

[0074] The cell culture incubator environment was set at 37°C and 5% (volume percentage) carbon dioxide (CO2).

[0075] In the following embodiments, the pH value of the phosphate buffer is 7.4, and the phosphate buffer is prepared by a disodium hydrogen phosphate aqueous solution and a sodium dihydrogen phosphate aqueous solution according to 81:19 (mass ratio), the disodium hydrogen phosphate aqueous solution is a mixture of anhydrous disodium hydrogen phosphate and water (the concentration of disodium hydrogen phosphate is 0.1 M), and the sodium dihydrogen phosphate aqueous solution is a mixture of anhydrous sodium dihydrogen phosphate and water (the concentration of sodium dihydrogen phosphate is 0.1 M).

[0076] In the following examples, the probe solution is a mixture of water, KCl, K3[Fe(CN)6] and K4[Fe(CN)6], the concentration of KCl in the probe solution is 0.1 M, and the concentrations of K3[Fe(CN)6] and K4[Fe(CN)6] are both 5 mM.

[0077] In the following embodiments, the differential pulse voltammetry (DPV) test is an oxidation DPV test or a reduction DPV test. According to the oxidation DPV test, an oxidation channel DPV curve is obtained, and the current value corresponding to the peak value of the oxidation channel DPV curve is the oxidation peak current. According to the reduction DPV test, a reduction channel DPV curve is obtained, and the current value corresponding to the peak value of the reduction channel DPV curve is the reduction peak current.

[0078] The differential pulse voltammetry (DPV) test includes: immersing the three-electrode system in the probe solution, starting the electrochemical workstation, and performing the differential pulse voltammetry (DPV) test, with a pulse amplitude of 0.05V, a pulse width of 0.05s, and a pulse period of 0.1s, wherein when performing the oxidation DPV test, the initial potential is set to -0.2V and the final potential is set to 0.6V; when performing the reduction DPV test, the initial potential is set to 0.6V and the final potential is set to -0.2V. The three-electrode system includes: a working electrode, a counter electrode, and a reference electrode, the counter electrode (CE) is a platinum sheet, and the reference electrode (RE) is a saturated calomel electrode (SCE).

[0079] Example 1

[0080] A method for preparing a gold-copper co-doped vertical graphene electrode (AuCu-), comprising the following steps:

[0081] Step 1: Use a roller mill to press the titanium wire into 30 mm, 1.6 mm wide, and 0.2 mm thick titanium bars (i.e., substrates), a total of 10 bars, use fine sandpaper to polish the surface of each titanium (Ti) bar to remove the oxide layer and make the surface rough, wipe the polished titanium bar with anhydrous ethanol and a dust-free cloth, then put it into a clean beaker, add ultrapure water, and use an ultrasonic cleaner to ultrasonically clean it at a frequency of 40KHz for 5 minutes. After the ultrasonic cleaning is completed, pour out the ultrapure water. , and add anhydrous ethanol, use anhydrous ethanol to ultrasonically clean for 5 minutes at a frequency of 40KHz, take out the titanium strips and place them in a clean culture dish to dry at room temperature for 10 minutes, add 10mL of the first solution to the culture dish containing the dried titanium strips, ensure that the first solution completely immerses 10 titanium strips, soak the dried titanium (Ti) strips in the first solution for 40 minutes, do not take out the titanium strips, and then directly use the photocatalytic method to convert Au in tetrachloroauric acid in the first solution onto the surface of the titanium strips. 3+ In situ reduction to gold nanoparticles, Cu in copper sulfate 2+In-situ reduction to copper nanoparticles, a gold-copper catalyst precursor layer is obtained on the surface of the titanium strip, and each titanium strip and the gold-copper catalyst precursor layer on its surface are used as the first substance, wherein the first solution is a mixture of tetrachloroauric acid (HAuCl4), copper sulfate (CuSO4) and water, and the ratio of gold in tetrachloroauric acid to copper in copper sulfate is 1:1 in terms of the amount of substance, the concentration of tetrachloroauric acid in the first solution is 0.1 mM, and the concentration of copper sulfate in the first solution is 0.1 mM. The photocatalytic method comprises: irradiating the first solution and the titanium strip immersed therein under a 375 W infrared lamp for 25 minutes to evaporate the first solution;

[0082] Step 2, open the reaction chamber of the hot wire CVD and diamond combined deposition system, use sandpaper to polish off the residual carbon powder and debris inside the reaction chamber, and wipe it clean with a dust-free cloth and anhydrous ethanol. After the reaction chamber is dry, hang 5 thin tantalum wires (filaments) in parallel on a copper rack, and the spacing between adjacent tantalum wires is 1 cm. Place the first substance obtained in step 1 on the sample stage (molybdenum stage) of the reaction chamber of the hot wire CVD and diamond combined deposition system, adjust the height of the sample stage so that the vertical distance between the first substance and the tantalum wire is 0.8 cm, seal the reaction chamber, turn on the water circulation, vacuum gauge and composite vacuum gauge in turn, start the mechanical pump, evacuate the reaction chamber to below 5 Pa, open the hydrogen bottle, introduce a small amount of hydrogen to remove the residual gas in the reaction chamber, wait for the pressure in the reaction chamber to drop below 5 Pa again, and continue to introduce methane (CH4) and hydrogen (H2) so that the volume ratio of methane and hydrogen is 18:300 (the flow rate of methane is 18 mL min -1 The flow rate of hydrogen is 300 mL min -1 ), when the pressure in the reaction chamber reaches 1700Pa, apply 120A AC power (voltage is 10V) to the filament in the reaction chamber through the AC filament power supply. When the pressure in the reaction chamber reaches 5100Pa, adjust the mechanical pump control valve to control the pressure at 5200Pa, and then continue to apply 120A AC power (voltage is 10V) for 30min under this pressure (5200Pa). After 30min, adjust the flow rate of methane and hydrogen so that the volume ratio of methane and hydrogen is 40:20 (adjust the flow rate of methane to 40mL min -1 , adjust the hydrogen flow rate to 20mL min -1 ), adjust the mechanical pump control valve to control the reaction chamber pressure at 400 Pa, use the bias system to apply a 6A DC bias (voltage of 30 V) between the sample stage and the filament for 6 minutes to form gold-copper co-doped vertical graphene nanosheets on the surface of the titanium strip to obtain gold-copper co-doped vertical graphene electrodes, wherein when the DC bias is applied, the DC bias and the AC power together keep the reaction chamber at 1000°C.

[0083] Example 1: Surface SEM of the gold-copper co-doped vertical graphene electrode prepared Figure 1 As shown, the cross-sectional SEM Figure 2 shown.

[0084] Comparative Example 1

[0085] A method for preparing a gold-doped vertical graphene electrode (Au-), which is substantially the same as that of Example 1, except that copper sulfate is not added to the first solution.

[0086] Comparative Example 2

[0087] A method for preparing a copper-doped vertical graphene electrode (Cu-), which is basically the same as that of Example 1, except that tetrachloroauric acid is not added to the first solution.

[0088] Comparative Example 3

[0089] A method for preparing a platinum-doped vertical graphene electrode (Pt-), which is basically the same as Example 1, except that the first solution is a mixture of hexachloroplatinic acid and water, and the concentration of hexachloroplatinic acid in the first solution is 0.1 mM.

[0090] Comparative Example 4

[0091] A method for preparing a gold-platinum co-doped vertical graphene electrode (AuPt-), which is basically the same as Example 1, except that: the first solution is a mixture of tetrachloroauric acid (HAuCl4), hexachloroplatinic acid and water, the ratio of gold in tetrachloroauric acid to platinum in hexachloroplatinic acid is 1:1, the concentration of tetrachloroauric acid in the first solution is 0.1 mM, and the concentration of hexachloroplatinic acid in the first solution is 0.1 mM.

[0092] Comparative Example 5

[0093] A method for preparing a copper-platinum co-doped vertical graphene electrode (CuPt-), which is basically the same as Example 1, except that: the first solution is a mixture of copper sulfate (CuSO4), hexachloroplatinic acid and water, and the ratio of copper in copper sulfate (CuSO4) to platinum in hexachloroplatinic acid is 1:1, the concentration of copper sulfate in the first solution is 0.1 mM, and the concentration of hexachloroplatinic acid in the first solution is 0.1 mM.

[0094] Comparative Example 6

[0095] A method for preparing a vertical graphene electrode (VG) is basically the same as that of Example 1, except that the first solution is water.

[0096] Example 2

[0097] Cyclic voltammetry (CV) test: Place the three-electrode system in the probe solution, start the electrochemical workstation, and perform cyclic voltammetry (CV) test. Set the initial potential to -0.6 V, the final potential to 1 V, and the scan rate to X, where the value range of X is 10 to 1000 mVs -1 , by setting different scan rates to obtain CV curves at different scan rates, where the scan rate is between 10 and 100 mVs -1 10mVs in the range -1 The scan rate is between 100 and 200 mVs. -1 20mVs in the range -1 The scan rate is between 200 and 1000 mVs. -1 50mVs in the range -1 The CV curves at different scanning rates are shown in Figure 3 As shown, Figure 3 a~g are the cyclic voltammetry scan rate change diagrams of Comparative Example 6, Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 1, Comparative Example 4 and Comparative Example 5, respectively. Figure 3 It can be seen that as the scan rate increases, the oxidation peak current increases and the reduction peak current decreases. Compared with comparative examples 1 to 6, the oxidation peak current of Example 1 is the largest at the same scan rate.

[0098] Electrochemical impedance spectroscopy test: Place the three-electrode system in the probe solution, start the electrochemical workstation, select the AC Impedance mode, and set the parameters as follows: open circuit voltage is 0.2 V, the frequency of the AC signal applied to the working electrode is 10 -2 ~10 6 Hz, and we get the Nyquist curve. The Nyquist curve is as follows Figure 4 As shown (when Example 1 and Comparative Examples 1 to 6 are used as working electrodes, each working electrode is tested 5 times continuously, that is, each working electrode obtains 5 Nyquist curves respectively), Figure 4 a to g are Nyquist curves of Comparative Example 6, Comparative Example 1, Comparative Example 2, Comparative Example 3, Example 1, Comparative Example 4 and Comparative Example 5, respectively. Figure 4 It can be seen that the semicircle diameter of Example 1 is significantly smaller than the semicircle diameters of Comparative Examples 1 to 6, indicating that the impedance of Example 1 is the smallest.

[0099] In the cyclic voltammetry (CV) test and the electrochemical impedance spectroscopy test, the three-electrode system includes a counter electrode, a reference electrode and a working electrode. The counter electrode is a platinum sheet, the reference electrode is a saturated calomel electrode, and the working electrode is one of the gold-copper co-doped vertical graphene electrode prepared in Example 1, the gold-doped vertical graphene electrode prepared in Comparative Example 1, the copper-doped vertical graphene electrode prepared in Comparative Example 2, the platinum-doped vertical graphene electrode prepared in Comparative Example 3, the gold-platinum co-doped vertical graphene electrode prepared in Comparative Example 4, the copper-platinum co-doped vertical graphene electrode prepared in Comparative Example 5, and the vertical graphene electrode prepared in Comparative Example 6.

[0100] based on Figure 3 The cyclic voltammetry scan rate change diagram and Figure 4 The Nyquist curve is used to calculate and analyze the electrochemical constants, and the electrochemical constants are the effective electroactive surface area, the electron transfer rate and the liquid-solid interface charge transfer resistance, wherein the effective electroactive surface area and the electron transfer rate are calculated respectively according to the Randles-Sevcik theoretical equation and the Laviron theoretical formula (refer to Chen Zehua. Preparation of gold platinum-boron doped graphene electrodes and their applications in immunosensors [D]. Tianjin University of Technology, 2023. DOI: 10.27360 / d.cnki.gtlgy.2023.000813.). Refer to Zhang X, Xu Y. The fabrication of hierarchical NiMoO4@Ni(OH)2nanocomposites and its electrochemical behavior used as supercapacitor electrode [J]. Inorganic Chemistry Communications, 2018, 87: 8-11. The liquid-solid interface charge transfer resistance is obtained. The electrochemical related constant data when Example 1 and Comparative Examples 1 to 6 are used as working electrodes are shown in the figure. Figure 5 As shown, Figure 5 a is the liquid-solid interface charge transfer resistance of Example 1 and Comparative Examples 1 to 6, Figure 5 b is the effective electroactive surface area of ​​Example 1 and Comparative Examples 1 to 6, Figure 5 c is the electron transfer rate of Example 1 and Comparative Examples 1 to 6. Figure 5 It can be seen that the gold-copper co-doped vertical graphene electrode prepared in Example 1 has a large effective electroactive surface area (0.48 cm 2 ), higher electron transfer rate (0.65cms -1 ) and smaller liquid-solid interface charge transfer resistance (8.14Ω), and has the best performance.

[0101] Example 3

[0102] A method for preparing a gold / gold-copper co-doped vertical graphene electrode comprises: immersing a three-electrode system in a tetrachloroauric acid aqueous solution (the three-electrode system comprises: a working electrode, a counter electrode and a reference electrode, the working electrode is the gold-copper co-doped vertical graphene electrode prepared in Example 1, the counter electrode (CE) is a platinum sheet, and the reference electrode (RE) is a saturated calomel electrode (SCE)), and using a multi-potential step electrochemical deposition method to deposit Au in the tetrachloroauric acid. 3+ Electrochemically reduced to gold nanoparticles and loaded on the surface of a gold-copper co-doped vertical graphene electrode (30 cycles of electrochemical deposition using a multi-potential step electrochemical deposition method, each cycle of electrodeposition includes: first at a potential of 2 V for 1 s, then at a potential of -2 V for 5 s), to obtain a gold / gold-copper co-doped vertical graphene electrode, wherein the concentration of tetrachloroauric acid in the tetrachloroauric acid aqueous solution is 1 mM.

[0103] Example 3: Surface SEM of the gold / gold-copper co-doped vertical graphene electrode prepared Figure 6 As shown, cross-sectional SEM Figure 7 shown.

[0104] The Raman spectrum of the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3 is as follows: Figure 8 As shown. Figure 8 The characteristic peaks G and 2D of graphene can be observed. The intensity ratio of the 2D peak to the G peak of the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3 (I 2D / I G ) is 1.27, and the half-peak width of the 2D peak is about 55.13 cm -1 , indicating that the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3 is composed of graphene nanosheets. The gold / gold-copper co-doped vertical graphene electrode prepared in Example 3 has a sharper D peak (1351cm -1 ), which is related to the vertical growth of graphene, exposing many atomic defects.

[0105] The X-ray photoelectron spectroscopy (XPS) of C1s, Au 4f, Cu 2p and O1s of the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3 was characterized. Fig. 9 .Depend on Fig. 9 It can be seen that in the Au 4f spectrum, the two peaks at 83.39eV and 88.06eV belong to Au 4f 7 / 2 and Au 4f 5 / 2 , which indicates that the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3 contains Au nanoparticles. For the Cu2p spectrum, the two peaks at 932.9 eV and 952.7 eV belong to Cu 2p 3 / 2 and Cu2p1 / 2 .

[0106] Example 4

[0107] The gold-copper co-doped vertical graphene electrode prepared in Example 1 and the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3 were used as working electrodes to perform differential pulse voltammetry (DPV) test (oxidation DPV test) to obtain an oxidation channel DPV curve. The oxidation peak current obtained according to the oxidation channel DPV curve of this embodiment is recorded as I 氧化 , the DPV curve of the oxidation channel is as follows Fig.10 A and Fig.10 As shown in b (when the working electrode is a gold-copper co-doped vertical graphene electrode, the oxidation channel DPV curve is Fig.10 In a, "AuCu-VG", when the working electrode is a gold / gold-copper co-doped vertical graphene electrode, the oxidation channel DPV curve is Fig.10 “Au / AuCu-VG” in b).

[0108] Example 5

[0109] The following Ab@gold / gold-copper co-doped vertical graphene electrode and the following Ab@gold-copper co-doped vertical graphene electrode were used as working electrodes to perform differential pulse voltammetry (DPV) test (oxidation DPV test) to obtain an oxidation channel DPV curve. The oxidation peak current obtained according to the oxidation channel DPV curve of this embodiment is recorded as I Ab-氧化 , the DPV curve of the oxidation channel is as follows Fig.10 A and Fig.10 As shown in b (when the working electrode is Ab@gold-copper co-doped vertical graphene electrode, the oxidation channel DPV curve is Fig.10 In a, when the working electrode is Ab@gold / gold-copper co-doped vertical graphene electrode, the DPV curve of the oxidation channel is Fig.10 “Ab@Au / AuCu-VG” in b).

[0110] The method for preparing the Ab@gold / gold-copper co-doped vertical graphene electrode is as follows: immerse the gold / gold-copper co-doped vertical graphene electrode prepared in Example 3 into a centrifuge tube containing 100 μL of CEA antibody (Ab) solution (the concentration of CEA antibody (Ab) in the CEA antibody (Ab) solution is 2500 ng·mL -1 ), the centrifuge tube was placed in an electric thermostatic incubator at 37°C for 75 min, and after incubation, the gold / gold-copper co-doped vertical graphene electrode was washed with phosphate buffered saline (PBS) to remove the excess CEA antibody (Ab) solution on the surface, and the electrode was placed at 37°C for 10 min to obtain the Ab@gold / gold-copper co-doped vertical graphene electrode. The concentration of CEA antibody (Ab) was 2500 ng·mL-1 The method of preparing CEA antibody (Ab) solution is as follows: phosphate buffered saline (PBS) is used to prepare 1 mg mL -1 Dilute the CEA antibody (Ab) solution.

[0111] The method for preparing the above-mentioned Ab@gold-copper co-doped vertical graphene electrode is basically the same as the “method for preparing Ab@gold / gold-copper co-doped vertical graphene electrode”, with the difference being that the “gold / gold-copper co-doped vertical graphene electrode prepared in Example 3” in the method for preparing Ab@gold / gold-copper co-doped vertical graphene electrode is replaced with the “gold-copper co-doped vertical graphene electrode prepared in Example 1”.

[0112] Fig.10 a is the DPV curve of the oxidation channel using the gold-copper co-doped vertical graphene electrode as the working electrode in Example 4 ( Fig.10 The DPV curves of the oxidation channel with Ab@gold-copper co-doped vertical graphene electrode as the working electrode in a) and Example 5 ( Fig.10 a in "Ab@AuCu-VG"), by Fig.10 From a, we can know the oxidation peak current I of the gold-copper co-doped vertical graphene electrode 氧化 =712.87μA, Ab@Oxidation peak current I of Au-Cu co-doped vertical graphene electrode Ab-氧化 =558.35μA; Fig.10 b is the DPV curve of the oxidation channel using the gold / gold-copper co-doped vertical graphene electrode as the working electrode in Example 4 ( Fig.10 The DPV curves of the oxidation channel with Ab@gold / gold-copper co-doped vertical graphene electrode as the working electrode in b) and Example 5 ( Fig.10 b) of “Ab@Au / AuCu-VG”, by Fig.10 From b, we can know that the oxidation peak current I of the gold / gold-copper co-doped vertical graphene electrode 氧化 =875.07μA, Ab@Oxidation peak current I of gold / gold-copper co-doped vertical graphene electrode Ab-氧化 =609.62μA. According to I 氧化 Subtract I Ab-氧化 It can be seen that the oxidation peak current difference between the gold / gold-copper co-doped vertical graphene electrode and the Ab@gold / gold-copper co-doped vertical graphene electrode is 265.45μA, which is larger than the oxidation peak current difference between the gold-copper co-doped vertical graphene electrode and the Ab@gold-copper co-doped vertical graphene electrode (154.52μA), that is, the gold / gold-copper co-doped vertical graphene electrode has a higher response ability to the tumor marker (CEA).

[0113] Example 6

[0114] A dual-channel gold-copper doped graphene electrochemical immunosensor was prepared. The method for preparing the dual-channel gold-copper doped graphene electrochemical immunosensor was as follows: 0.03 g of bovine serum albumin powder was weighed in a cillin bottle, and 2 mL of phosphate buffer (PBS) was added to prepare a bovine serum albumin (BSA) solution with a concentration of 1.5 wt %. 100 μL of the bovine serum albumin (BSA) solution was taken with a pipette and transferred to a centrifuge tube, and the Ab@gold / gold-copper co-doped vertical graphene electrode prepared in Example 5 was immersed in the bovine serum albumin (BSA) solution in the centrifuge tube, and the centrifuge tube was placed in an electric thermostatic incubator at a temperature of 37° C. for 15 min, and the Ab@gold / gold-copper co-doped vertical graphene electrode was washed with phosphate buffer (PBS), and the excess BSA solution on the surface was removed, and the electrode was allowed to stand at 37° C. for 10 min to obtain a dual-channel gold-copper doped graphene electrochemical immunosensor.

[0115] The dual-channel gold-copper doped graphene electrochemical immunosensor was used as a working electrode for DPV test (oxidation DPV test) to obtain an oxidation channel DPV curve. The oxidation peak current obtained according to the oxidation channel DPV curve was recorded as I1, I1 = 453.78 μA.

[0116] The dual-channel gold-copper doped graphene electrochemical immunosensor was used as a working electrode for DPV test (reduction DPV test) to obtain a reduction channel DPV curve. The reduction peak current obtained according to the reduction channel DPV curve was recorded as I1, I1 = -447.91 μA.

[0117] The proteins contained in bovine serum albumin can bind to nonspecific sites on the surface of gold nanoparticles and occupy these sites, thereby reducing the binding of antigens to nonspecific sites on the surface of gold nanoparticles during subsequent incubation, reducing nonspecific adsorption, and increasing the accuracy of test results.

[0118] Example 7

[0119] A method for detecting the concentration of an antigen, comprising:

[0120] 1) Prepare N standard solutions, N = 16, each standard solution is a mixture of antigen and phosphate buffer (PBS), the antigen (Ag) concentration in the N standard solutions is different, each standard solution is used as a sample solution to calculate the ΔI of each sample solution; the antigen is carcinoembryonic antigen (CEA), and the concentration of carcinoembryonic antigen (CEA) in the 16 standard solutions is 0.001 pg·mL -1 , 0.005pg·mL -1 , 0.01pg·mL -1 , 0.05pg·mL -1 , 0.1pg·mL -1 , 0.5pg·mL-1 , 1pg·mL -1 , 5pg·mL -1 , 10pg·mL -1 , 50pg·mL -1 , 100pg·mL -1 , 500pg·mL -1 , 1000pg·mL -1 , 5000pg·mL -1 , 10000pg·mL -1 and 30000pg·mL -1 Each standard solution is 1 mg·mL -1 The carcinoembryonic antigen (CEA) solution was diluted with phosphate buffer.

[0121] 2) Establishing a coordinate system, using the lg value of the antigen concentration and ΔI as the abscissa and ordinate respectively, substituting the lg value and ΔI of the antigen (Ag) concentration of N portions of the standard solution into the coordinate system, and fitting to obtain a linear regression equation;

[0122] 3) 1 mg·mL -1 The carcinoembryonic antigen (CEA) solution was diluted with phosphate buffer to a CEA concentration of 0.1 pg mL -1 The solution to be tested is used as the sample solution, the ΔI of the solution to be tested is calculated, and the ΔI of the solution to be tested is substituted into the linear regression equation to obtain the lg value of the antigen concentration in the solution to be tested, and then the antigen concentration in the solution to be tested is calculated.

[0123] The method for calculating ΔI of each sample solution includes:

[0124] ① The dual-channel gold-copper-doped graphene electrochemical immunosensor in Example 6 was immersed in 100 μL of the sample solution, incubated in a 37°C electric thermostatic incubator for 15 min, washed with phosphate buffered saline (PBS), and allowed to stand at 37°C for 10 min to obtain a sensor. The sensor was subjected to a differential pulse voltammetry (DPV) test, and the current value corresponding to the peak value of the DPV curve was obtained as I2;

[0125] ② Calculate ΔI of the sample solution, ΔI=I1-I2, where I1 is the current value corresponding to the peak value of the DPV curve obtained by performing differential pulse voltammetry (DPV) test on the dual-channel gold-copper doped graphene electrochemical immunosensor in Example 6.

[0126] The above antigen concentration detection method is used for operation, wherein the differential pulse voltammetry (DPV) test is an oxidation DPV test. When the differential pulse voltammetry (DPV) test is an oxidation DPV test, the oxidation channel DPV curve obtained by the differential pulse voltammetry (DPV) test of 16 standard solutions is as follows: Fig.11 As shown (opening downward). I1 = 453.78 μA. The linear regression equation (oxidation channel linear regression equation) is as follows Fig.12 As shown. pa =5.98log c1+24.60, log c1 is the lg value of antigen concentration, ΔI pa The current value I2 corresponding to the peak value of the oxidation channel DPV curve of the test solution is 435.07 μA. The ΔI of the test solution = 453.78 μA-435.07; Substitute the ΔI of the test solution into the linear regression equation to obtain the lg value of the antigen concentration in the test solution, and then calculate that the antigen concentration in the test solution is 0.104 pg mL -1 , and the deviation from the actual value of the antigen concentration in the test solution is within 5%.

[0127] The above-mentioned antigen concentration detection method is used for operation, wherein the differential pulse voltammetry (DPV) test is a reduction DPV test. When the differential pulse voltammetry (DPV) test is a reduction DPV test, the reduction channel DPV curve obtained by the differential pulse voltammetry (DPV) test of 16 standard solutions is as follows: Fig.11 As shown (opening upward). I1 = -447.91 μA. The linear regression equation (reduction channel linear regression equation) is as follows Fig.12 As shown. pc =-5.88log c2–23.15, log c2 is the lg value of antigen concentration, ΔI pc The current value I2 corresponding to the peak value of the reduction channel DPV curve of the test solution is -430.57μA. The ΔI of the test solution = -447.91μA + 430.57μA. Substitute the ΔI of the test solution into the linear regression equation to obtain the lg value of the antigen concentration in the test solution, and then calculate the antigen concentration in the test solution to be 0.103pg mL -1 , and the deviation from the actual value of the antigen concentration in the test solution is within 5%.

[0128] Fig.11 “E / V(vs.SCE)” indicates the test potential of differential pulse voltammetry, and “c[CEA] / pg mL -1 " indicates the carcinoembryonic antigen (CEA) concentration in the standard solution (Balnk indicates the carcinoembryonic antigen concentration is 0 pg·mL -1, equivalent to a dual-channel gold-copper-doped graphene electrochemical immunosensor), "I / μA" represents the current. Fig.11 It can be seen that as the concentration of carcinoembryonic antigen (CEA) in the standard solution increases, the oxidation peak current decreases continuously (e.g., 0.001 pg·mL -1 When the oxidation peak current is 448.3μA; 50pg·mL -1 When the oxidation peak current is 418.94μA; 30000pg·mL -1 When the oxidation peak current was 402.54 μA, it indicated that the DPV test was responsive to the CEA concentration.

[0129] like Fig.12 As shown, no matter the differential pulse voltammetry (DPV) test is an oxidation DPV test or a reduction DPV test, the fitting degree of the linear regression equation is above 0.99, which is suitable for detecting the carcinoembryonic antigen (CEA) concentration.

[0130] In Example 1, the gold-copper co-doped vertical graphene electrode ( Fig.13 "AuCu-") in Example 3, gold / gold-copper co-doped vertical graphene electrode ( Fig.13 "AuAu-" in Example 5, Ab@gold / gold-copper co-doped vertical graphene electrode ( Fig.13 "Ab-") in Example 6, the dual-channel gold-copper-doped graphene electrochemical immunosensor ( Fig.13 "BSA-") and the sensor in Example 7 ( Fig.13 The 3D image of "Ag-") under an atomic force microscope is as follows Fig.13 As shown. Fig.13 It can be seen that after the deposition of gold (Au) nanoparticles on the gold-copper co-doped vertical graphene electrode, the roughness of the gold / gold-copper co-doped vertical graphene electrode was improved due to the rapid edge deposition effect of the vertical graphene nanosheets. After incubation with CEA antibody (Ab), the pores of the Ab@gold / gold-copper co-doped vertical graphene electrode were reduced and shallow; while after incubation with BSA, the surface of the dual-channel gold-copper doped graphene electrochemical immunosensor became flat. It can be seen that during the layer-by-layer incubation of CEA antibody (Ab) and bovine serum albumin (BSA), these protein molecules filled the pores between the graphene sheets. After incubation with carcinoembryonic antigen (CEA), the roughness of the sensor increased, indicating that antibody-antigen immune complexes were formed at specific sites.

[0131] Example 8

[0132] The initial MCF-7 tumor cell concentration was 34 cells μL. -1 , 170 cells μL -1 , 306 cells μL -1442 cells μL -1 and 578 cells μL -1 The MCF-7 tumor cell fluid (MCF-7 tumor cell fluid includes: MCF-7 tumor cells and cell culture medium, MCF-7 tumor cells grow by climbing the wall), the MCF-7 tumor cell fluids of different concentrations were placed in a 37°C cell culture incubator for 12 hours, and 100 μL of liquid was extracted from the upper layer of the MCF-7 tumor cell fluids of different concentrations. The liquid contained carcinoembryonic antigen (CEA) released by MCF-7 tumor cells, and the extracted 100 μL of liquid was diluted 1000 times with PBS to obtain MCF-7 tumor cell dilutions. For the MCF-7 tumor cell dilutions obtained from the MCF-7 tumor cell fluids of different MCF-7 tumor cell concentrations, 100 μL of each MCF-7 tumor cell dilution was taken as a test solution. The solution to be tested is used as the sample solution in the antigen concentration detection method, the ΔI of the solution to be tested is calculated, and the ΔI of the solution to be tested is substituted into the linear regression equation to obtain the lg value of the antigen concentration in the solution to be tested, and then the antigen concentration in the solution to be tested is calculated. The method for calculating ΔI is the same as the method for calculating ΔI of each sample solution in Example 7.

[0133] When the differential pulse voltammetry (DPV) test is an oxidation DPV test, the oxidation channel DPV curves of the five test solutions are as follows: Fig.14 as shown (opening facing downwards). Fig.14 The local enlarged image of the DPV curve of the oxidation channel shows that the initial MCF-7 tumor cell concentration is 34 cells μL -1 , 170 cells μL -1 , 306 cells μL -1 442 cells μL -1 and 578 cells μL -1 The oxidation peak current (I2) under the conditions of 423.32μA, 422.53μA, 421.99μA, 421.6μA and 421.34μA, respectively. The I2 of the five solutions to be tested is 423.32μA, 422.53μA, 421.99μA, 421.6μA and 421.34μA. The I1 of the five solutions to be tested is 456.77μA. According to ΔI=I1-I2, the ΔI of each solution to be tested is calculated, and the ΔI of each solution to be tested is substituted into the linear regression equation ΔI in Example 7. pa =5.98log c1+24.60, where c1 is the antigen concentration in the test solution.

[0134] When the differential pulse voltammetry (DPV) test is a reduction DPV test, the reduction channel DPV curves of the five test solutions are as follows: Fig.14 as shown (opening facing upward). Fig.14 The local enlarged image of the reduction channel DPV curve shows that the initial MCF-7 tumor cell concentration is 34 cells μL -1 , 170 cells μL -1 , 306 cells μL -1 442 cells μL -1 and 578 cells μL -1 The reduction peak current (I2) under the conditions of ΔI=I1-I2 was -419.31μA, -418.57μA, -417.99μA, -417.58μA and -417.39μA, respectively. The I1 of the five test solutions was -451.14μA. The I2 of the five standard solutions was -419.31μA, -418.57μA, -417.99μA, -417.58μA and -417.39μA. The ΔI of each test solution was calculated according to ΔI=I1-I2, and the ΔI of each test solution was substituted into the linear regression equation ΔI in Example 7. pc =-5.88log c2–23.15, where c2 is the antigen concentration in the test solution.

[0135] The CEA concentration of the above test solutions was determined according to the instructions of the commercial ELISA kits, and the CEA concentration obtained by the commercial ELISA kit was recorded as c3.

[0136] Since the MCF-7 tumor cell dilution was obtained by diluting PBS 1000 times, c1, c2 and c3 were multiplied by 1000, and c1 was multiplied by 1000 as Fig.15 Medium Ox , c2 multiplied by 1000 is Fig.15 Medium Re , c3 multiplied by 1000 is Fig.15 Medium ELISA .Depend on Fig.15 It can be seen that c Ox 、c Re With c ELISA The results are basically consistent, indicating that the CEA concentration obtained by the linear regression equation of the oxidation channel and the CEA concentration obtained by the linear regression equation of the reduction channel are basically consistent with the CEA concentration obtained by the commercial ELISA kit.

[0137] Since the concentrations of CEA and coexisting interferents (such as cellular metabolites) in the MCF-7 tumor cell dilution are different, it is unclear which of the oxidation channel and reduction channel detection results is more accurate, so measuring the average of the two is conducive to providing more reliable detection data. Ox +c Re ) / 2=c mean (c mean cOx and c Re The mean of mean like Fig.15 shown.

[0138] The dual-channel gold-copper doped graphene electrochemical immunosensor of the present invention has good stability and low detection limit. According to the long-term stability test method mentioned in Zheng S, Li M, Li H, et al. Sandwich-type electrochemical immunosensor for carcinoembryonic antigen detection based on the cooperation of a gold-vertical graphene electrode and gold@silica-methylene blue[J]. Journal of Materials Chemistry B, 2020, 8. DOI: 10.1039 / C9TB01803D., the dual-channel gold-copper doped graphene electrochemical immunosensor of the present invention is stored at 4°C. After testing, after 82 days, the oxidation peak current of the dual-channel gold-copper doped graphene electrochemical immunosensor still maintains 93.1% of the initial oxidation peak current, and the reduction peak current still maintains 93.2% of the initial reduction peak current. Although the activity of BSA incubated on the surface of the dual-channel gold-copper doped graphene electrochemical immunosensor changes over time, it has little effect on ΔI.

[0139] Similarly, according to the detection limit calculation method in Zheng S, Li M, Li H, et al. Sandwich-type electrochemical immunosensor for carcinoembryonic antigen detection based on the cooperation of a gold-vertical graphene electrode and gold@silica-methylene blue[J]. Journal of Materials Chemistry B, 2020, 8. DOI: 10.1039 / C9TB01803D., it can be calculated that the detection limit of the dual-channel gold-copper doped graphene electrochemical immunosensor for CEA of the present invention is 0.28 fg mL -1 .

[0140] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A gold / gold-copper co-doped vertical graphene electrode, characterized in that: include: A gold-copper co-doped vertical graphene electrode and gold nanoparticles loaded on the surface of the gold-copper co-doped vertical graphene electrode. The gold-copper co-doped vertical graphene electrode comprises a substrate and gold-copper co-doped vertical graphene nanosheets grown on the surface of the substrate.

2. The gold / gold-copper co-doped vertical graphene electrode according to claim 1, characterized in that: The substrate is made of titanium or tantalum.

3. A method for preparing a gold / gold-copper co-doped vertical graphene electrode, characterized in that: include: The gold-copper co-doped vertical graphene electrode was used as the working electrode in the three-electrode system. The three-electrode system was immersed in an aqueous solution of tetrachloroauric acid. The Au in the tetrachloroauric acid was deposited by a multi-potential step electrochemical deposition method. 3+ The gold nanoparticles are electrochemically reduced to gold nanoparticles and loaded on the surface of a gold-copper co-doped vertical graphene electrode to obtain a gold / gold-copper co-doped vertical graphene electrode. The gold-copper co-doped vertical graphene electrode comprises a substrate and gold-copper co-doped vertical graphene nanosheets grown on the surface of the substrate.

4. The preparation method according to claim 3, characterized in that: The concentration of tetrachloroauric acid in the tetrachloroauric acid aqueous solution is 0.8-1.2 mM; the multi-potential step electrochemical deposition method has 5-100 cycles of electrodeposition, and each cycle of electrodeposition includes: first, a potential of 1-2 V for 1-2 s, and then a potential of -2--1 V for 4-6 s.

5. A method for preparing a gold-copper co-doped vertical graphene electrode, characterized in that: The following steps are involved: Step 1: immerse the substrate in the first solution, and use the photocatalytic method to catalyze Au in tetrachloroauric acid in the first solution on the surface of the substrate. 3+ In situ reduction to gold nanoparticles, Cu in copper sulfate 2+ In-situ reduction to copper nanoparticles, obtaining a gold-copper catalyst precursor layer on the surface of the substrate, the substrate and the gold-copper catalyst precursor layer on its surface are used as the first substance, wherein the first solution comprises: tetrachloroauric acid, copper sulfate and water, the ratio of gold in tetrachloroauric acid to copper in copper sulfate is 1:(0.8-1.2) in terms of the amount of substance, and the concentration of tetrachloroauric acid in the first solution is 0.05-0.15 mM; Step 2, placing the first material obtained in step 1 on a sample stage of a reaction chamber in a hot filament CVD and diamond combined deposition system, sealing the reaction chamber, evacuating the reaction chamber to below 5 Pa, continuously introducing methane and hydrogen so that the volume ratio of methane to hydrogen is (15-20): (250-350), applying 110-130A alternating current to the filament in the reaction chamber when the pressure in the reaction chamber reaches 1600-1700Pa, and continuing to apply 110-130A alternating current under the pressure when the pressure in the reaction chamber reaches 4500-5500Pa. Apply an alternating current of 30A for 30 to 40 minutes, then adjust the flow rate of methane and hydrogen so that the volume ratio of methane and hydrogen is (30 to 50): (15 to 25), and when the pressure of the reaction chamber reaches 300 to 500 Pa, apply a direct current bias of 5 to 8A between the sample stage and the filament for 3 to 10 minutes to form gold-copper co-doped vertical graphene nanosheets on the surface of the substrate to obtain a gold-copper co-doped vertical graphene electrode, wherein when the direct current bias is applied, the direct current bias and the alternating current jointly keep the reaction chamber at 900 to 1100° C.

6. A dual-channel gold-copper doped graphene electrochemical immunosensor, characterized in that: include: Gold / gold-copper co-doped vertical graphene electrode and bovine serum albumin and antibody incubated on the gold / gold-copper co-doped vertical graphene electrode.

7. A method for preparing the dual-channel gold-copper doped graphene electrochemical immunosensor according to claim 6, characterized in that: The following steps are involved: S1, incubating the antibody on the gold / gold-copper co-doped vertical graphene electrode to obtain Ab@gold / gold-copper co-doped vertical graphene electrode; S2, incubation of bovine serum albumin on Ab@Au / AuCu co-doped vertical graphene electrode.

8. A method for detecting the concentration of an antigen, characterized in that: include: 1) Prepare N standard solutions, each of which is a mixture of antigen and phosphate buffer. The antigen concentrations in the N standard solutions are different. Each standard solution is used as a sample solution to calculate ΔI. 2) Establishing a coordinate system, taking the lg value of antigen concentration and ΔI as the horizontal and vertical coordinates, substituting the lg value and ΔI of antigen concentration of N portions of standard solution into the coordinate system, and fitting to obtain a linear regression equation; 3) The solution to be tested is used as the sample solution, the ΔI of the solution to be tested is calculated, and the ΔI of the solution to be tested is substituted into the linear regression equation to obtain the lg value of the antigen concentration in the solution to be tested, and then the antigen concentration in the solution to be tested is calculated.

9. The concentration detection method according to claim 8, characterized in that: The method for calculating ΔI for each sample solution includes: ① Incubating antigens on the dual-channel gold-copper-doped graphene electrochemical immunosensor with a sample solution to obtain a sensor; performing a differential pulse voltammetry test on the sensor to obtain a current value corresponding to the peak value of the DPV curve as I2; ② Calculate the ΔI of the sample solution, ΔI = I1-I2, where I1 is the current value corresponding to the peak value of the DPV curve obtained by differential pulse voltammetry test of the dual-channel gold-copper doped graphene electrochemical immunosensor.

10. A method for detecting the concentration of a tumor marker, characterized in that: include: Based on the oxidation DPV test, the antigen concentration in the test solution is obtained according to the antigen concentration detection method as c1. Based on the reduction DPV test, the antigen concentration in the test solution is obtained according to the antigen concentration detection method as c2. The value of 0.5 (c1 + c2) is used as the tumor marker concentration.

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