Electrochemical immunosensor for simultaneous detection of two neuroendocrine tumor markers

Abstract

The application discloses an electrochemical immunosensor for simultaneously detecting two neuroendocrine tumor markers, and belongs to the technical field of electrochemical biosensors.The sensor uses two parallel glassy carbon electrodes as working electrodes, and the electrode surfaces are sequentially modified with metal ion functionalized porous magnesium silicate / gold nanoparticle / polyethylene glycol / chitosan composite materials and recognition antibodies.When specific binding occurs between the markers and the antibodies, the simultaneous detection of CgA and CgB is realized by recording the electric signals distinguishable by two metal ions through differential pulse voltammetry, and the more the markers react with the antibodies, the lower the electric signals are.The electrochemical immunosensor has the advantages of high sensitivity, low detection limit, can make up for the limitations of single markers, and improves the accuracy of neuroendocrine tumor diagnosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of electrochemical biosensing, and provides an electrochemical immunosensor for simultaneously detecting neuroendocrine tumor markers, i.e., simultaneously detecting two neuroendocrine tumor (NETs) markers chromogranin A (CgA) and chromogranin B (CgB). BACKGROUND

[0002] Neuroendocrine tumors (NETs) are a group of rare tumors originating from neuroendocrine cells. In recent years, the incidence of NETs has been continuously increasing worldwide. NETs are usually asymptomatic in the early stage, and 60-80% of NETs patients are diagnosed as advanced stage. NETs such as rectal and pancreatic NETs are prone to misdiagnosis and mistreatment in clinic. Therefore, effective tumor marker screening and early diagnosis of NETs are crucial for improving the treatment effect of patients.

[0003] At present, serum chromogranin A (CgA) is the most commonly used marker in NETs, which can be used to assist diagnosis, guide treatment and evaluate efficacy. The existing serum CgA detection method has low sensitivity and is not sensitive to early diagnosis. In addition, oral proton pump inhibitors (PPIs) and patients with renal dysfunction, inflammatory bowel disease and other pancreatic diseases, etc. CgA appears false elevation. Chromogranin B (CgB) is a secretory protein of the same family as CgA, which can be used as a biomarker for NETs. The positive rate of CgB in rectal and pancreatic NETs is higher than that of CgA, and it is not affected by factors such as PPIs, thereby making up for the shortcomings of insufficient sensitivity of CgA. Therefore, simultaneous detection of serum CgA and CgB is of great significance for improving the accuracy of clinical diagnosis of NETs, but there is currently no biosensor for simultaneous detection of CgA and CgB. SUMMARY

[0004] The purpose of the present application is to provide an electrochemical immunosensor for simultaneously detecting two neuroendocrine tumor markers, i.e., an electrochemical immunosensor for simultaneously detecting two NETs markers CgA and CgB, and a preparation method of the electrochemical immunosensor for simultaneously detecting CgA and CgB.

[0005] The electrochemical immunosensor for simultaneously detecting two NETs markers CgA and CgB of the present application comprises a working electrode composed of two parallel glassy carbon electrodes, and the surface of the glassy carbon electrode is sequentially modified with a metal ion functionalized porous magnesium silicate / gold nanoparticle / polyethylene glycol / chitosan (PMS-M 2+ / AuNPs / PEG / CS) composite material and an antibody.

[0006] The metal ions adsorbed in the composite material modified on the surface of one glassy carbon electrode are copper ions (Cu 2+The modified antibody is a CgA antibody, used to identify and detect CgA; the other glassy carbon electrode surface is modified with a composite material in which lead ions (Pb) are adsorbed. 2+ The modified antibody is a CgB antibody, used to identify and detect CgB. Cu 2+ and Pb 2+ It has distinct differential pulse voltammetry (DPV) peak potentials, and its peak currents are used as signal outputs for the simultaneous electrochemical detection of CgA and CgB.

[0007] The method for manufacturing this sensor includes the following steps:

[0008] (1) Metal ion functionalized porous magnesium silicate (PMS-M) 2+ Preparation of )

[0009] Add 8–12 mg of PMS to 10–30 mL of copper nitrate or lead nitrate solution, wherein the mass ratio of PMS to copper nitrate (or lead nitrate) is 1:(0.5–3). After ultrasonic dispersion, stir at 20–25 °C for 6–10 h, then centrifuge and wash, and dry in an oven at 55–65 °C to obtain PMS-Cu. 2+ or PMS-Pb 2+ .

[0010] (2) PMS-M 2+ Preparation of / AuNPs / PEG / CS

[0011] Solution A: Concentration of 1–3 mg / mL -1 PMS-Cu 2+ or PMS-Pb 2+ Solution A: A mixed solution containing 0.4–0.6 wt.% polyethylene glycol (PEG), 0.4–0.6 wt.% chitosan (CS), and 0.4–0.6 mM gold nanoparticles (AuNPs). Solutions A and B were mixed at a volume ratio of 1:(0.5–2) and ultrasonically dispersed.

[0012] (3) Preparation of the working electrode of the sensor

[0013] 1) Add 5–10 μL of PMS-Cu 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS solution was dropped onto the surfaces of two clean glassy carbon electrodes and dried at 20-25°C.

[0014] 2) Use PMS-Cu in step 1) 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS modified glassy carbon electrodes were respectively used with 5-10 μL of 8-12 μg mL solutions. -1 The CgA and CgB antibody solutions were incubated overnight at 3–5°C, then rinsed with deionized water and stored at 3–5°C for later use.

[0015] The PMS-M 2+ The PMS in the sample was prepared by the following method:

[0016] Magnesium nitrate hexahydrate was dissolved in a mixed solvent to prepare a solution with a concentration of 8–12 mg / L. -1 A magnesium nitrate solution is prepared, wherein the mixed solvent is a mixture of ethanol and water, and the ethanol accounts for 96-98% of the volume of the mixed solvent. The concentration is prepared to be 0.2-0.25 mg / mL. -1 A sodium silicate aqueous solution was prepared. Under stirring conditions, sodium silicate solution was added dropwise to magnesium nitrate solution at a volume ratio of 1:(9-12), and the mixture was stirred for 1-5 minutes to obtain a white slurry. The resulting white slurry was transferred to a hydrothermal reactor and heated to 160-180°C for 18-30 hours. After the reaction was completed, the mixture was allowed to cool naturally to room temperature, centrifuged, and washed with deionized water and ethanol until neutral. The precipitate obtained by centrifugation was dried in an oven at 55-65°C to obtain PMS.

[0017] The sensor is prepared by the following method, specifically the AuNPs prepared in step (2):

[0018] Under stirring, 90–110 mL of a 0.8–1.2 mM chloroauric acid solution was heated to boiling and refluxed. Then, 8–12 mL of a 35–40 mM sodium citrate solution was rapidly added to the above solution. When the solution turned wine-red, it was boiled for another 10–20 minutes under stirring to obtain an AuNPs dispersion.

[0019] This sensor is used for the simultaneous detection of CgA and CgB, and the steps are as follows:

[0020] (1) Add 0.1 pg mL -1 ~100ng mL -1 A series of CgA and CgB standard solutions of different concentrations were dropped onto the glassy carbon electrode modified with CgA and CgB antibodies as described in claim 1, and incubated at 36.5–37.5°C for 30–90 min, then rinsed with deionized water and dried.

[0021] (2) An electrochemical workstation was used for testing with a three-electrode system. Two parallel modified glassy carbon electrodes were used as working electrodes, Pt wire as the counter electrode, and saturated Ag / AgCl as the reference electrode. Acetic acid buffer solution with pH 4.5–5.5 was used as the test substrate. The detection was performed using the DPV method, with a scanning voltage range of -0.7–0.3 V. Cu before and after immobilization of the biomarker protein was recorded. 2+ and Pb 2+ The peak current values ​​were used for quantitative detection of CgA and CgB, respectively. The more markers that react with the antibody, the lower the current value.

[0022] (3) According to Cu 2+ and Pb 2+ The working curve was constructed by analyzing the relationship between the change in current and the corresponding concentrations of CgA and CgB standard solutions. Figure 4 The differential pulse voltammograms of the electrochemical immunosensor prepared in Example 1 for simultaneously detecting different concentrations of CgA and CgB are shown. Figure 5 and Figure 6 The figures show the operating curves for the electrochemical immunosensor prepared in Example 1 detecting CgA and CgB, respectively. As shown in the figures, the peak current decreases with increasing concentrations of CgA and CgB in the sample solution. The operating curves indicate a good linear relationship between the peak current and the logarithm of the target antigen concentration. The linear range for CgA detection is 0.1 pg / mL. -1 Up to 100 ng / mL -1 The detection limit is 5.3 fg / mL. -1 (S / N = 3). The detection limit for CgB (within the same concentration range as CgA) is 2.1 fg / mL. -1 (S / N = 3)

[0023] (4) Replace the CgA and CgB standard solutions with the test sample solution and test the test sample according to the above method. Based on the working curve, obtain the concentration of CgA and CgB in the test sample.

[0024] The beneficial effects of this invention are:

[0025] (1) The electrochemical immunosensor constructed in this invention can be used for the simultaneous detection of two NETs markers, CgA and CgB.

[0026] (2) This invention introduces PMS into the fabrication of an electrochemical immunosensor. PMS, as a three-dimensional flower-like porous silicate material with a high specific surface area, is effective for Cu... 2+ and Pb 2+ It has significant adsorption capacity and can serve as an effective carrier for adsorbing metal ions, enabling signal amplification and highly sensitive detection.

[0027] (3) This invention uses PMS-M2+ / AuNPs / PEG / CS composite material as a sensing interface. The combination of PEG and CS not only serves to immobilize PMS-M 2+ AuNPs, but also can synergistically enhance the anti-fouling ability of the sensing interface, improving the specificity, selectivity and stability of the sensor. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Schematic diagram of simultaneous detection of the electrochemical immunosensor of the present application.

[0029] Figure 2 Scanning electron microscope image of PMS-Cu 2+ prepared in Example 1.

[0030] Figure 3 Scanning electron microscope image of PMS-Pb 2+ prepared in Example 1.

[0031] Figure 4 Differential pulse voltammogram of the electrochemical immunosensor prepared in Example 1 for simultaneous detection of different concentrations of CgA and CgB.

[0032] Figure 5 Working curve graph of the electrochemical immunosensor prepared in Example 1 for detection of CgA.

[0033] Figure 6 Working curve graph of the electrochemical immunosensor prepared in Example 1 for detection of CgB. DETAILED DESCRIPTION

[0034] The present application will be further described below in conjunction with the drawings and specific embodiments, but the present application is not limited to the following examples.

[0035] As introduced in the background art, there is a need to develop an analytical method for simultaneous detection of serum CgA and CgB, and for this purpose, the present patent provides an electrochemical immunosensor for simultaneous detection of CgA and CgB.

[0036] The schematic diagram of simultaneous detection of the electrochemical immunosensor is shown in Figure 1 Cu 2+ and Pb 2+ have distinguishable DPV peak potentials at about 0.03 V and -0.47 V (relative to a saturated Ag / AgCl electrode), respectively, and thus are used as electroactive substances. In order to amplify the signal, PMS is used as an effective carrier for adsorbing metal ions, and PMS is a two-dimensional layered silicate material with a high specific surface area, which has a significant adsorption capacity for Cu 2+ and Pb 2+ . By immobilizing metal ions Cu 2+ and Pb2+ PMS-Cu 2+ and PMS-Pb 2+ composite materials to achieve high sensitivity signal output. Then PMS-M 2+ / AuNPs / PEG / CS mixed layer is used as a sensing interface, PEG and CS are not only used to fix PMS-M 2+ and AuNPs, but also the composite of the two can synergistically enhance the antifouling ability of the sensing interface. The capture antibodies of CgA and CgB are fixed on the biosensing interface PMS-Cu 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS, realizing the construction of a label-free electrochemical immunosensor. Once specific binding occurs between NETs biomarkers and antibodies, simultaneous quantitative detection of CgA and CgB can be realized by recording the changes in the electrochemical signals of Cu 2+ and Pb 2+ , the more biomarkers that react with the antibodies, the lower the electrochemical signal.

[0037] In order to enable those skilled in the art to more clearly understand the technical solutions of the present disclosure, the technical solutions of the present disclosure will be described in detail below in combination with specific embodiments.

[0038] Embodiment 1

[0039] 1. Preparation of a simultaneous detection CgA and CgB electrochemical immunosensor

[0040] (1) Preparation of PMS-M 2+

[0041] 10 mg of PMS was added to 20 mL of copper nitrate or lead nitrate solution, and the mass ratio of PMS to copper nitrate (or lead nitrate) was 1:2. After ultrasonic dispersion of the solution, it was stirred at 22°C for 8 h, then centrifuged and washed, and dried in a 60°C oven to prepare PMS-Cu 2+ or PMS-Pb 2+ . Figure 2 The scanning electron microscope image of the prepared PMS-Cu 2+ , Figure 3 The scanning electron microscope image of the prepared PMS-Pb 2+ .

[0042] (2) Preparation of PMS-M 2+ / AuNPs / PEG / CS

[0043] Solution A: PMS-Cu -1 with a concentration of 2 mg / mL 2+ ​or PMS-Pb 2+ Solution; Solution B: mixed solution containing 0.5wt.% PEG, 0.5wt.% CS and 0.5mM AuNPs. Solution A and B were mixed according to the volume ratio of 1:1 and ultrasonic dispersion.

[0044] (3) Preparation of sensor working electrode

[0045] 1) 6μL PMS-Cu 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS solution was dropped on the surface of two clean glassy carbon electrodes respectively, and dried at 22℃;

[0046] 2) The PMS-Cu 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS modified glassy carbon electrodes were respectively incubated with 6μL CgA and CgB antibody solution with a concentration of 10μg mL -1 at 4℃ overnight, then rinsed with deionized water and stored at 4℃ for standby.

[0047] 2, Preparation of PMS

[0048] Magnesium nitrate hexahydrate was dissolved in a mixed solvent, a magnesium nitrate solution with a concentration of 10mg L -1 was prepared, and the mixed solvent was a mixed solvent of ethanol and water, in which the volume percentage of ethanol in the mixed solvent was 97%. A sodium silicate aqueous solution with a concentration of 0.23mg mL -1 was prepared. Under stirring conditions, the sodium silicate solution was added dropwise into the magnesium nitrate solution according to the volume ratio of 1:11, and stirred for 3min to obtain a white slurry. The obtained white slurry was transferred to a hydrothermal reactor, heated to 170℃, and reacted for 24h. After the reaction was completed, it was naturally cooled to room temperature, centrifuged, and washed with deionized water and ethanol until neutral, and the obtained precipitate was dried in a 60℃ oven, to obtain PMS.

[0049] 3, Preparation of AuNPs

[0050] Under stirring conditions, 100mL of chloroauric acid solution with a concentration of 1.0mM was heated to boiling reflux, and then 10mL of sodium citrate solution with a concentration of 38.8mM was quickly added into the above solution. When the solution color changed to wine red, boiling was continued under stirring conditions for 15min to obtain AuNPs dispersion.

[0051] 4, Simultaneous detection of CgA and CgB

[0052] (1) 0.1 pg mL -1 ~ 100 ng mL -1 A series of different concentrations of CgA and CgB standard solutions were added dropwise to the glassy carbon electrodes modified with CgA and CgB antibodies, respectively, and incubated at 37°C for 60 min, washed with deionized water and dried;

[0053] (2) The electrochemical workstation was used to test in a three-electrode system, two parallel glassy carbon electrodes were used as working electrodes, a Pt wire was used as a counter electrode, a saturated Ag / AgCl was used as a reference electrode, a pH 5.0 acetic acid buffer solution was used as a test base, and a DPV method was used to detect, the peak current values of Cu 2+ and Pb 2+ before and after the fixed marker protein were recorded for quantitative detection of CgA and CgB, and the more antibodies reacted with the marker, the lower the current value.

[0054] (3) According to the relationship between the change difference of the current of Cu 2+ and Pb 2+ and the concentration of the corresponding CgA and CgB standard solution, a working curve was prepared.

[0055] (4) The sample solution to be tested was used instead of the CgA and CgB standard solution, and the sample to be tested was detected according to the above method, and the concentration of CgA and CgB in the sample to be tested was obtained according to the working curve.

[0056] 5. Actual blood sample detection

[0057] A recovery experiment was performed by a standard addition method to verify the feasibility of the electrochemical immunosensor of the present application in clinical diagnosis. Human serum samples were taken, centrifuged, and the supernatant was detected by the constructed sensor to detect the content of CgA and CgB in the human serum sample, and then a certain concentration of CgA and CgB standard solution was added to the human serum, and the recovery rate of CgA and CgB in the sample was calculated by the ratio of the detected recovery value to the added amount, and the results are shown in Table 1.

[0058] Table 1 Detection results of CgA and CgB in human serum samples

[0059]

[0060] n in the table is the number of parallel detections.

[0061] As shown in Table 1, the relative standard deviation of the detection results is less than 5.0%, and the recovery rate is between 96.8% and 105%, indicating that the electrochemical immunosensor of the present application can be used for simultaneous detection of CgA and CgB in human serum, and the detection results are accurate and reliable.

[0062] Example 2

[0063] 1. Preparation of an electrochemical immunosensor for simultaneous detection of CgA and CgB

[0064] (1) Preparation of PMS-M 2+

[0065] 8 mg of PMS was added into 10 mL of copper nitrate or lead nitrate solution, with the mass ratio of PMS to copper nitrate (or lead nitrate) being 1:0.5, the solution was ultrasonically dispersed and stirred at 20°C for 6 h, then centrifuged and washed, and dried in an oven at 55°C to obtain PMS-Cu 2+ or PMS-Pb 2+ .

[0066] (2) Preparation of PMS-M 2+ / AuNPs / PEG / CS

[0067] Solution A: PMS-Cu -1 or PMS-Pb 2+ solution with a concentration of 1 mg mL 2+ ; Solution B: mixed solution containing 0.4 wt.% PEG, 0.4 wt.% CS and 0.4 mM AuNPs. Solution A and B were mixed in a volume ratio of 1:0.5 and ultrasonically dispersed.

[0068] (3) Preparation of the working electrode of the sensor

[0069] 1) 5 μL of PMS-Cu 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS solution were dropped onto the surface of two clean glassy carbon electrodes respectively and dried at 20°C;

[0070] 2) The glassy carbon electrodes modified with PMS-Cu 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS in step 1) were respectively incubated with 5 μL of CgA and CgB antibody solution with a concentration of 8 μg mL -1 at 3°C overnight, then rinsed with deionized water and stored at 3°C for standby.

[0071] 2. Preparation of PMS

[0072] Magnesium nitrate hexahydrate was dissolved in a mixed solvent to prepare a magnesium nitrate solution with a concentration of 8 mg L -1 , the mixed solvent being a mixed solvent of ethanol and water, with the volume percentage of ethanol in the mixed solvent being 96%. A solution with a concentration of 0.2 mg mL -1 was prepared.Aqueous solution of sodium silicate. Under stirring condition, the sodium silicate solution was added dropwise into the magnesium nitrate solution with a volume ratio of 1 :9, and stirred for 1 min to obtain a white slurry. The obtained white slurry was transferred into a hydrothermal reactor, heated to 160 °C, and reacted for 18 h. After the reaction, it was naturally cooled to room temperature, centrifuged, washed with deionized water and ethanol until neutral, and the obtained precipitate was dried in a 55 °C oven to obtain PMS.

[0073] 3. Preparation of AuNPs

[0074] Under stirring condition, 90 mL of HAuCl4 solution with a concentration of 0.8 mM was heated to boiling reflux, and then 8 mL of Na3C6H5O7 solution with a concentration of 35 mM was quickly added into the above solution. When the solution color changed to wine red, boiling was continued under stirring condition for 10 min to obtain AuNPs dispersion.

[0075] 4. Simultaneous detection of CgA and CgB

[0076] The same as the simultaneous detection of CgA and CgB in Example 1, except that the incubation conditions of CgA and CgB were incubated at 36.5 °C for 30 min, and the DPV test base solution was acetic acid buffer solution with pH 4.5.

[0077] Example 3

[0078] 1. Preparation of an electrochemical immunosensor for simultaneous detection of CgA and CgB

[0079] (1) Preparation of PMS-M 2+

[0080] 12 mg of PMS was added into 30 mL of copper nitrate or lead nitrate solution, with a mass ratio of PMS to copper nitrate (or lead nitrate) of 1 :3, the solution was ultrasonically dispersed, then stirred at 25 °C for 10 h, centrifuged and washed, and dried in a 65 °C oven to prepare PMS-Cu 2+ or PMS-Pb 2+ .

[0081] (2) Preparation of PMS-M 2+ / AuNPs / PEG / CS

[0082] Solution A: PMS-Cu -1 or PMS-Pb 2+ solution with a concentration of 3 mg mL 2+ ; Solution B: mixed solution containing 0.6 wt.% PEG, 0.6 wt.% CS and 0.6 mM AuNPs. Solution A and B were mixed with a volume ratio of 1 :2, and ultrasonically dispersed. ​

[0083] (3) Preparation of sensor working electrode

[0084] 1) 10 μL PMS-Cu 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS solution was dropped onto the surface of two clean glassy carbon electrodes respectively, and dried at 25 °C;

[0085] 2) The PMS-Cu 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS modified glassy carbon electrodes were respectively incubated with 10 μL of CgA and CgB antibody solution with a concentration of 12 μg mL -1 at 5 °C overnight, then rinsed with deionized water and stored at 5 °C for standby.

[0086] 2. Preparation of PMS

[0087] Magnesium nitrate hexahydrate was dissolved in a mixed solvent, a magnesium nitrate solution with a concentration of 12 mg L -1 was prepared, and the mixed solvent was a mixture of ethanol and water, in which the volume percentage of ethanol in the mixed solvent was 98%. A sodium silicate aqueous solution with a concentration of 0.25 mg mL -1 was prepared. Under stirring conditions, the sodium silicate solution was added dropwise into the magnesium nitrate solution at a volume ratio of 1:12, and stirred for 5 min to obtain a white slurry. The obtained white slurry was transferred into a hydrothermal reactor, heated to 180 °C, and reacted for 30 h. After the reaction was completed, it was naturally cooled to room temperature, centrifuged, and washed with deionized water and ethanol until neutral. The obtained precipitate was dried in a 65 °C oven, and PMS was obtained.

[0088] 3. Preparation of AuNPs

[0089] Under stirring conditions, 110 mL of chloroauric acid solution with a concentration of 1.2 mM was heated to boiling reflux, and then 12 mL of sodium citrate solution with a concentration of 40 mM was quickly added into the above solution. When the color of the solution changed to wine red, boiling was continued under stirring conditions for 20 min to obtain an AuNPs dispersion.

[0090] 4. Simultaneous detection of CgA and CgB

[0091] The same as the simultaneous detection of CgA and CgB in Example 1, except that the incubation conditions of CgA and CgB were incubated at 37.5 °C for 90 min, and the DPV test base solution was an acetic acid buffer solution with pH 5.5.

[0092] The above embodiments describe the details of the present application, but the present application is not limited thereto. Any improvement and various simple modifications to the technical solutions of the present application within the technical concept of the present application all belong to the protection scope of the present application.

Claims

1. A method for preparing an electrochemical immunosensor for simultaneously detecting two neuroendocrine tumor markers, characterized in that, Includes the following steps: (1) Metal ion functionalized porous magnesium silicate (PMS-M) 2+ Preparation of ) Add 8–12 mg of PMS to 10–30 mL of copper nitrate or lead nitrate solution, wherein the mass ratio of PMS to copper nitrate, or PMS to lead nitrate, is 1:(0.5–3). After ultrasonic dispersion, stir at 20–25 °C for 6–10 h, then centrifuge and wash, and dry in an oven at 55–65 °C to obtain PMS-Cu. 2+ or PMS-Pb 2+ ; (2) PMS-M 2+ Preparation of / AuNPs / PEG / CS Solution A: Concentration of 1–3 mg / mL -1 PMS-Cu 2+ or PMS-Pb 2+ Solution A: Solution B: A mixed solution containing 0.4–0.6 wt.% polyethylene glycol (PEG), 0.4–0.6 wt.% chitosan (CS), and 0.4–0.6 mM gold nanoparticles (AuNPs); Solutions A and B are mixed at a volume ratio of 1:(0.5–2) and ultrasonically dispersed. (3) Fabrication of the sensor working electrode 1) Add 5-10 μL PMS-Cu 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS solution was dropped onto the surfaces of two clean glassy carbon electrodes and dried at 20-25°C. 2) Use PMS-Cu in step 1) 2+ / AuNPs / PEG / CS and PMS-Pb 2+ / AuNPs / PEG / CS modified glassy carbon electrodes were respectively used with 5-10 μL of 8-12 μg mL solutions. -1 The CgA and CgB antibody solutions were incubated overnight at 3–5°C, then rinsed with deionized water and stored at 3–5°C for later use.

2. The electrochemical immunosensor obtained by the preparation method according to claim 1, characterized in that, The working electrode consists of two glassy carbon electrodes connected in parallel, the surfaces of which are sequentially modified with metal ion-functionalized porous magnesium silicate / gold nanoparticles / polyethylene glycol / chitosan (PMS-M). 2+ / AuNPs / PEG / CS) composite materials and antibodies; The metal ions adsorbed in a composite material modified with a glassy carbon electrode surface are copper ions (Cu). 2+ The modified antibody is a CgA antibody, used to identify and detect CgA; the metal ions adsorbed in the composite material modified on the surface of the other glassy carbon electrode are lead ions (Pb). 2+ The modified antibody is a CgB antibody, used to identify and detect CgB; wherein, Cu 2+ and Pb 2+ The differential pulse voltammetry (DPV) peak potentials are distinguishable, and their peak currents are used as signal outputs for the simultaneous electrochemical detection of CgA and CgB, respectively.

3. The electrochemical immunosensor according to claim 2, characterized in that, The steps for simultaneous detection of CgA and CgB using this sensor are as follows: (1) Take 0.1 pg mL -1 ~100ng mL -1 A series of CgA and CgB standard solutions of different concentrations were dropped onto glassy carbon electrodes modified with CgA and CgB antibodies, and incubated at 36.5–37.5 °C for 30–90 min, then rinsed with deionized water and dried. (2) An electrochemical workstation was used for testing with a three-electrode system. Two parallel modified glassy carbon electrodes were used as working electrodes, Pt wire as the counter electrode, and saturated Ag / AgCl as the reference electrode. Acetic acid buffer solution with pH 4.5–5.5 was used as the test substrate. The detection was performed using the DPV method, with a scanning voltage range of -0.7–0.3 V. Cu before and after immobilization of the biomarker protein was recorded. 2+ and Pb 2+ The peak current values ​​were used for quantitative detection of CgA and CgB, respectively. The more markers that react with the antibody, the lower the current value. (3) According to Cu 2+ and Pb 2+ The relationship between the change in current and the corresponding concentrations of CgA and CgB standard solutions was investigated to create a working curve. (4) Replace the CgA and CgB standard solutions with the test sample solution and test the test sample according to the above method. Based on the working curve, obtain the concentration of CgA and CgB in the test sample.

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