Preparation method and application of electrochemical biosensor for detecting HER-2

Abstract

The invention belongs to the technical field of rapid detection of sensors and tumor markers, and relates to a preparation method and application of an electrochemical biosensor for detecting HER-2. The MB (at) ZnMOF-AMWCNTs nano composite material is used for preparing a sensor; mB (at) ZnMOF is used as an electroactive indicator, has abundant carboxyl groups, and can promote biomolecule conjugation; electron transfer on the surface of the electrode is enhanced through the AMWCNTs, so that the conductivity of the biosensor is improved; the HER-2 in serum is detected through the specific reaction of the antigen and the antibody, the detection method is simple, the result is accurate, and the efficiency is high; the method has the performance of rapid DPV response, high sensitivity and low-limit detection. The preparation method of the electrochemical biosensor is novel, simple to operate and low in cost, and a new auxiliary platform is provided for detection of tumor biomarkers.

Description

Technical field:

[0001] The present invention belongs to the technical field of sensors and rapid detection of tumor markers, and particularly relates to a preparation method and application of an electrochemical biosensor for detecting human epidermal growth factor receptor 2 (HER-2). Background technology:

[0002] Breast cancer (BC), as the most common cancer in women, has posed a major threat to women's health and survival. It is worth noting that one of the most lethal BC subtypes is human epidermal growth factor receptor 2 (HER-2) positive BC. And about 15-20% of patients are characterized by HER-2 gene amplification or protein overexpression, which leads to more aggressive tumors and unfavorable clinical prognosis. HER-2 is a transmembrane tyrosine kinase receptor whose extracellular domain can be shed into the blood. In BC patients, the concentration of HER-2 is generally higher than 15.0 ng mL -1 Therefore, accurate assessment of HER-2 levels can help clinicians choose the best treatment strategy and significantly improve the prognosis of HER-2-positive BC patients.

[0003] To date, HER-2 has been assayed using various methods, such as immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and electrochemiluminescence analysis (ECLA). However, many traditional analytical techniques have inherent limitations. For example, IHC and FISH always require professional operation and observer experience to interpret complex result data, which greatly affects the accuracy of the test. In addition, NGS is expensive and time-consuming, while ECLA requires a large amount of instrument costs. In addition, ELISA may produce false positive or false negative results.

[0004] In recent years, electrochemical immunosensors have demonstrated broad application potential in medical testing, environmental monitoring, and food safety due to their high sensitivity, fast response, portability, and low cost. Furthermore, the incorporation of nanomaterials into electrochemical systems can significantly enhance immunosensor performance. Metal-organic frameworks (MOFs) are novel crystalline molecular materials composed of metal ions / clusters linked by organic ligands via coordination bonds. Compared to three-dimensional (3D) cubic MOFs, two-dimensional (2D) MOF nanosheets not only retain the intrinsic characteristics of their bulk counterparts but also possess larger surface area, numerous accessible metal active sites, strong adsorption capacity, and excellent mechanical flexibility, contributing to improved performance in sensing-related applications. These properties make MOFs promising for broad application in electrochemical sensors. However, to date, research on electrochemical biosensors for detecting HER-2 in breast cancer is relatively limited. Summary of the invention:

[0005] To address the shortcomings of existing technologies, the present invention provides a method for preparing an electrochemical biosensor for detecting HER-2 and its application. The electrochemical biosensor, fabricated using MB@ZnMOF and AMWCNTs, exhibits high sensitivity, good specificity, a wide linear range, and a low detection limit, and can be applied to the detection of HER-2 in serum.

[0006] To achieve the above objectives, the present invention provides a method for preparing methylene blue @ ZnMOF. In the present invention, MB@ZnMOF is used as an electroactive indicator for electrochemical biosensors and is prepared by a bottom-up ultrasonic method. The preparation steps are as follows:

[0007] 40-100 mg ZnO and 20-70 mg 1,3,5-benzenetricarboxylic acid were simultaneously added to 10-80 mL of ethanol aqueous solution, and then 10-50 mg of methylene blue was added to the mixture and stirred. The mixture was then ultrasonically treated at room temperature for 1-6 hours to obtain a blue composite material, which was washed with DMF and deionized water in sequence and dried at 10-80°C for 6-18 hours to obtain MB@ZnMOF crystals.

[0008] The present invention also provides an electrochemical biosensor for detecting HER-2, which has a structure in which the surface of a base electrode is covered with an MB@ZnMOF-AMWCNTs composite material; a 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide activator is fixed on the surface of the MB@ZnMOF-AMWCNTs composite material; a HER-2 antibody is connected to the surface of the activator, and the antibody can undergo a specific recognition reaction with the target HER-2 antigen.

[0009] The present invention also provides a method for preparing the electrochemical biosensor for detecting HER-2, comprising the following steps:

[0010] (1) Electrode pretreatment: grinding, polishing and ultrasonic cleaning of the base electrode;

[0011] (2) Synthesis of MB@ZnMOF-AMWCNTs composite material: 0.5-10 μL of an aqueous solution of amino-modified multi-walled carbon nanotubes (0.1-8 mg / mL) and 0.5-10 μL of an aqueous solution of MB@ZnMOF (0.1-10 mg / mL) were sequentially added dropwise to the electrode surface obtained in step (1), and the mixture was irradiated under an infrared lamp until dry, until the modified material was completely bound to the electrode surface, and then washed with water and dried at room temperature;

[0012] (3) Covalently linking HER2 antibody: 0.5-10 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution with a concentration of 1-40 mg / mL and 0.5-10 μL of 1-40 mg / mL N-hydroxysuccinimide aqueous solution were sequentially added to the electrode obtained in step (2) and allowed to stand at room temperature for 0.5-4 hours; the residual activator was washed with distilled water; then, 0.5-10 μL of HER2 antibody (1-20 μg / mL Ab) solution was added dropwise to the electrode surface and allowed to stand at 0-20°C for 6-24 hours to allow Ab and MB@ZnMO to fully connect, and then washed with PBS solution and dried at room temperature;

[0013] (4) Blocking nonspecific sites: adding 0.1-10 wt% BSA aqueous solution, leaving it at room temperature for 10-90 min to block the unbound sites, washing it with PBS solution, and drying it at room temperature to obtain an electrochemical biosensor for detecting HER-2.

[0014] Preferably, the substrate electrode is a glassy carbon electrode.

[0015] In step (1) of the present invention, the base electrode is polished with Al2O3 powders having particle sizes of 0.1-2 μm and 0.01-0.1 μm, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 1-10 minutes. Preferably, the base electrode is polished with Al2O3 powders having particle sizes of 0.5 μm and 0.03 μm, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 5 minutes.

[0016] Preferably, in step (2), the concentration of the aqueous solution of aminated multi-walled carbon nanotubes is 0.1-8 mg / mL, and the drop volume is 0.5-10 μL; the concentration of the aqueous solution of MB@ZnMOF is 0.1-10 mg / mL, and the drop volume is 0.5-10 μL.

[0017] The concentration of Ab in step (3) of the present invention is 1-20 μg / mL, preferably, the concentration of Ab is 8-10 μg / mL; more preferably, the concentration of Ab is 8 μg / mL.

[0018] The present invention also provides an electrochemical biosensor prepared by the above preparation method and its application in quantitative detection of HER-2. The detection is for non-diagnostic purposes.

[0019] The application method is as follows: after the HER-2 solution to be tested is dripped onto the above-mentioned biosensor, the HER-2 protein will be incubated with the antibody pre-modified on the electrode surface to form an antigen-antibody immune complex; by monitoring the current signal of the biosensor in a solution containing 1.0mM K3[Fe(CN)6], the concentration of the HER-2 solution to be tested is calculated using a standard relationship curve.

[0020] The incubation temperature of the present invention is 10-70°C, and the incubation time is 10-90 minutes; preferably, the incubation temperature is 20-60°C, and the incubation time is 20-60 minutes; more preferably, the incubation temperature is 37°C, and the incubation time is 40 minutes.

[0021] The standard relationship curve of the present invention is obtained by electrochemically detecting a HER-2 standard solution with a concentration of 0.1-200 ng / mL using the electrochemical biosensor to obtain the peak current intensity corresponding to each concentration of the HER-2 standard solution, and then fitting the result with the logarithm of the concentration of the HER-2 standard solution as the abscissa and the peak current intensity as the ordinate. The standard relationship curve is I (μA) = -2.191lgC HER-2 +20.552. The detection limit was 0.05 ng / mL.

[0022] The abbreviations of the technical terms in the present invention are as follows:

[0023] Antibody: Ab; human epidermal growth factor receptor 2: HER-2; zinc-based metal organic framework: ZnMOF; amino-modified carbon nanotubes: AMWCNTs; methylene blue: MB; 1,3,5-benzenetricarboxylic acid: H3BTC; glassy carbon electrode: GCE; bovine serum albumin: BSA; differential pulse voltammetry: DPV; cyclic voltammetry: CV.

[0024] Compared with existing technologies, this invention utilizes a novel label-free electrochemical sensor fabricated using MB@ZnMOF-AMWCNTs nanocomposite materials. MB@ZnMOF, an electroactive indicator, possesses abundant carboxyl groups, which facilitate biomolecule conjugation. AMWCNTs enhance electron transfer at the electrode surface, thereby increasing the biosensor's conductivity. HER-2 is detected in serum through a specific reaction between antigen and antibody, resulting in a simple, accurate, and efficient detection method. The biosensor exhibits rapid DPV response, high sensitivity, and a low detection limit. This electrochemical biosensor boasts a novel fabrication method, simple operation, and low cost, providing a new auxiliary platform for the detection of tumor biomarkers. Description of the drawings:

[0025] Figure 1 This is the XPS graph of MB@ZnMOF.

[0026] Figure 2 The current responses of different modified electrodes in 1.0 mM K3[Fe(CN)6] substrate solution.

[0027] Figure 3 Graph showing the effects of Ab concentration (A), incubation temperature (B), and incubation time (C) on the electrochemical signal.

[0028] Figure 4 This is a linear relationship diagram between the peak current intensity of the electrochemical sensor used to measure HER-2 and the HER-2 concentration. Specific implementation method:

[0029] The technical solution of the present invention is further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0030] Example 1:

[0031] This embodiment relates to a method for preparing the electroactive indicator MB@ZnMOF, and the specific steps include:

[0032] 81 mg ZnO and 50.0 mg H3BTC were added simultaneously to 40 mL of ethanol-water solution (the volume ratio of ethanol to deionized water was 1:1), and then 20 mg MB (methylene blue) was added to the mixture and stirred. It was then ultrasonically treated at room temperature for 4 h to obtain a blue composite material, which was washed with DMF and deionized water in sequence and dried at 60 °C for 12 h to obtain MB@ZnMOF crystals.

[0033] MB@ZnMOF was characterized by XPS. Figure 1 shown.

[0034] Example 2:

[0035] This embodiment relates to a method for preparing the electroactive indicator MB@ZnMOF, and the specific steps include:

[0036] 40 mg of ZnO and 20 mg of 1,3,5-benzenetricarboxylic acid were added simultaneously to 10 mL of ethanol-water solution, and then 10 mg of methylene blue was added to the mixture and stirred. The mixture was then ultrasonically treated at room temperature for 1 h to obtain a blue composite material, which was washed with DMF and deionized water in sequence and dried at 10 °C for 18 h to obtain MB@ZnMOF crystals.

[0037] Example 3:

[0038] This embodiment relates to a method for preparing the electroactive indicator MB@ZnMOF, and the specific steps include:

[0039] 100 mg ZnO and 70 mg 1,3,5-benzenetricarboxylic acid were added simultaneously to 80 mL of ethanol aqueous solution, and then 50 mg methylene blue was added to the mixture and stirred. The mixture was then ultrasonically treated at room temperature for 6 h to obtain a blue composite material, which was washed with DMF and deionized water in sequence and dried at 80 °C for 6 h to obtain MB@ZnMOF crystals.

[0040] Example 4:

[0041] This embodiment relates to a method for preparing an electrochemical biosensor for detecting HER-2, and the specific steps are as follows:

[0042] (1) Electrode pretreatment: The glassy carbon electrode was ground, polished, and ultrasonically cleaned. Specifically, the electrode was polished with 0.5 μm and 0.03 μm Al2O3 powders, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 5 min.

[0043] (2) Synthesis of MB@ZnMOF-AMWCNTs composites: 5 μL AMWCNTs (3.0 mg mL -1 ) aqueous solution and 5 μL of MB@ZnMOF (5.0 mg mL -1 ) aqueous solution is added dropwise to the electrode surface obtained in step (1), dried under infrared light until the synthesized MB@ZnMOF-AMWCNTs composite material is completely bonded to the electrode surface, washed with water, and dried at room temperature to obtain MB@ZnMOF-AMWCNTs / GCE;

[0044] (3) Covalently linking HER-2 antibody: 5.0 μL of 20.0 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) aqueous solution and 5.0 μL of 10.0 mg / mL NHS (N-hydroxysuccinimide) aqueous solution were sequentially added to the electrode obtained in step (2) and allowed to stand at room temperature for 2 hours; the residual activator was washed with distilled water; then, 5 μL of HER-2 antibody (8 μg / mL, Ab) solution was added dropwise to the electrode surface and allowed to stand at 4°C for 12 hours to allow Ab and the EDC / NHS activated material on the MB@ZnMOF surface to be fully linked through amide bonds, then washed with PBS solution and dried at room temperature to obtain MB@ZnMOF-AMWCNTs / Ab / GCE;

[0045] (4) Blocking nonspecific sites: 1 wt% BSA aqueous solution was added dropwise, and the mixture was allowed to stand at room temperature for 60 min to block the unbound sites. The mixture was washed with PBS solution and dried at room temperature to obtain MB@ZnMOF-AMWCNTs / Ab / BSA / GCE, which is an electrochemical biosensor for detecting HER-2.

[0046] (5) Detection of HER-2: (a) Testing the standard relationship curve: Different concentrations of antigen HER-2 protein were added to the biosensor and incubated at 37°C for 40 minutes to obtain the sensor electrode to be tested MB@ZnMOF-AMWCNTs / Ab / BSA / HER-2 / GCE. The sensor electrode to be tested was placed in a solution containing 1.0 mM K3[Fe(CN)6] and the peak current signal of the biosensor was monitored by differential pulse voltammetry to obtain the peak current intensity corresponding to different HER-2 concentrations, and the standard relationship curve was drawn; (b) 5 μL of the HER-2 protein to be tested was added to the biosensor and incubated at 37°C for 40 minutes. The unbound antigen was washed with PBS solution, and the current signal of the biosensor was monitored by differential pulse voltammetry in a solution containing 1.0 mM K3[Fe(CN)6]. The concentration of the target antigen HER-2 protein was calculated according to the standard relationship curve.

[0047] The electrochemical biosensor prepared in this example is used to detect HER-2 based on the principle that the Ab antibody modified on the sensor surface can specifically bind to the HER-2 protein to form an antigen-antibody immune complex. By monitoring the change in current intensity after the immune complex is formed and using a standard curve of the relationship between current intensity and HER-2 specific antigen sample concentration, the concentration of the HER-2 specific antigen sample to be tested can be determined.

[0048] The glassy carbon electrode obtained in each step above was placed in a solution containing 1.0 mM K3[Fe(CN)6] and cyclic voltammetry was performed at a rate of 0.1 V / s. Figure 2 As shown, the bare GCE exhibits a pair of distinct redox peaks, attributed to the reversible redox reaction of ferrocyanide ions. Compared to the bare GCE, the CV response significantly increases when MB@ZnMOF-AMWCNTs are modified on the electrode, demonstrating its excellent electron transfer capability as a substrate material. Subsequently, as antibodies, bovine serum albumin (BSA), and HER-2 bind to the prepared GCE surface, the redox peak current gradually decreases due to the insulating properties of the protein molecules. These results during the electrode modification process demonstrate the successful fabrication of the electrochemical biosensor.

[0049] Example 5:

[0050] This embodiment relates to a method for preparing an electrochemical biosensor for detecting HER-2, and the specific steps are as follows:

[0051] (1) Electrode pretreatment: The base electrode was ground, polished, and ultrasonically cleaned. Specifically, the base electrode was polished with 0.1 μm and 0.01 μm Al2O3 powders, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 1 min.

[0052] (2) Synthesis of MB@ZnMOF-AMWCNTs composite material: 0.5 μL of an aqueous solution of amino-modified multi-walled carbon nanotubes (0.1 mg / mL) and 0.5 μL of an aqueous solution of MB@ZnMOF (0.1 mg / mL) were sequentially added dropwise to the electrode surface obtained in step (1), and the mixture was irradiated under an infrared lamp until it was completely bound to the electrode surface. The mixture was then washed with water and dried at room temperature.

[0053] (3) Covalently linking HER2 antibody: 0.5 μL of a 1 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution and 1 μL of a 1 mg / mL N-hydroxysuccinimide aqueous solution were sequentially added to the electrode obtained in step (2) and allowed to stand at room temperature for 0.5 h. The residual activator was washed with distilled water. Subsequently, 2 μL of a HER2 antibody (1 μg / mL Ab) solution was added to the electrode surface and allowed to stand at 0°C for 6 h to allow Ab and MB@ZnMO to fully connect. The electrode was then washed with PBS solution and dried at room temperature.

[0054] (4) Blocking nonspecific sites: add 0.1 wt% BSA aqueous solution, let it stand at room temperature for 10 min to block the unbound sites, wash with PBS solution, and dry at room temperature to obtain an electrochemical biosensor for detecting HER-2.

[0055] Example 6:

[0056] This embodiment relates to a method for preparing an electrochemical biosensor for detecting HER-2, and the specific steps are as follows:

[0057] (1) Electrode pretreatment: The base electrode was ground, polished, and ultrasonically cleaned. Specifically, the base electrode was polished with 2 μm and 0.1 μm Al2O3 powders, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 10 min.

[0058] (2) Synthesis of MB@ZnMOF-AMWCNTs composite material: 10 μL of an aqueous solution of amino-modified multi-walled carbon nanotubes (8 mg / mL) and 8 μL of an aqueous solution of MB@ZnMOF (10 mg / mL) were sequentially added dropwise to the electrode surface obtained in step (1), and the mixture was irradiated under an infrared lamp until it was completely bound to the electrode surface. The mixture was then washed with water and dried at room temperature.

[0059] (3) Covalently linking HER2 antibody: 10 μL of a 40 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution and 10 μL of a 40 mg / mL N-hydroxysuccinimide aqueous solution were sequentially added to the electrode obtained in step (2) and allowed to stand at room temperature for 4 hours; the residual activator was washed with distilled water; then, 10 μL of a 20 μg / mL HER2 antibody solution was added to the electrode surface and allowed to stand at 20°C for 24 hours to allow Ab and MB@ZnMO to fully connect, and then washed with PBS solution and dried at room temperature;

[0060] (4) Blocking nonspecific sites: 10 wt% BSA aqueous solution was added dropwise and allowed to stand at room temperature for 90 min to block unbound sites. The solution was then washed with PBS solution and dried at room temperature to obtain an electrochemical biosensor for detecting HER-2.

[0061] Example 7:

[0062] This embodiment relates to a method for preparing an electrochemical biosensor for detecting HER-2, and the specific steps are as follows:

[0063] (1) Electrode pretreatment: The base electrode was ground, polished, and ultrasonically cleaned. Specifically, the base electrode was polished with 1 μm and 0.05 μm Al2O3 powders, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 4 min.

[0064] (2) Synthesis of MB@ZnMOF-AMWCNTs composite material: 2 μL of amino-modified multi-walled carbon nanotube aqueous solution (1 mg / mL) and 10 μL of MB@ZnMOF (1 mg / mL) aqueous solution were sequentially added dropwise to the electrode surface obtained in step (1), and irradiated under an infrared lamp until dry. After the modified material was completely bound to the electrode surface, the mixture was washed with water and dried at room temperature.

[0065] (3) Covalently linking HER2 antibody: 0.5 μL of a 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution and 0.5 μL of a 10 mg / mL N-hydroxysuccinimide aqueous solution were sequentially added to the electrode obtained in step (2) and allowed to stand at room temperature for 2 h. The residual activator was washed with distilled water. Subsequently, 0.5 μL of a HER2 antibody (5 μg / mL Ab) solution was added dropwise to the electrode surface and allowed to stand at 10°C for 20 h to allow Ab and MB@ZnMO to fully connect. The electrode was then washed with PBS solution and dried at room temperature.

[0066] (4) Blocking nonspecific sites: Add 5 wt% BSA aqueous solution and let it stand at room temperature for 50 min to block the unbound sites. Wash with PBS solution and dry at room temperature to obtain an electrochemical biosensor for detecting HER-2.

[0067] Example 8:

[0068] This example involves an experiment on the effects of Ab concentration, HER-2 antigen antibody incubation time, and incubation temperature on the sensitivity and catalytic performance of the biosensor. Biosensors with different Ab concentrations (6-10 μg / mL) were prepared in 1.0 mM K3[Fe(CN)6] solution according to the method of Example 4. The effect of Ab concentration on the biosensor signal response was tested using DPV (differential pulse voltammetry). The results are shown in Figure 2. Figure 3 As shown in A. Figure 3 In A, as the Ab concentration continued to increase from 6 μg / mL to 8 μg / mL, the current difference (ΔI = initial peak current value - peak current value after reaction) reached its maximum at 8 μg / mL. Subsequently, the antibody concentration continued to increase, while ΔI tended to stabilize, indicating that the optimal concentration of Ab was 8 μg / mL. In addition, the incubation temperature of HER-2 antigen antibody also affects the sensing performance of the biosensor. Therefore, the MB@ZnMOF-AMWCNTs / Ab / BSA / GCE prepared in Example 4 was incubated with HER-2 protein (concentration of 10 ng / mL) at 20-60°C for 40 minutes to detect the effect on the current response. The results are shown in Figure 1. Figure 3 As shown in B. Figure 3 B shows that the current value is close to the highest at 37°C. As the temperature continues to increase, ΔI decreases. This is because the high temperature damages the immune complex. Therefore, 37°C was selected as the optimal incubation temperature. Finally, the incubation time of HER-2 protein binding to the capture Ab was also studied (including 20, 30, 40, 50 and 60 minutes). The results are shown in Figure 2. Figure 3 As shown in Figure C, 40 minutes of incubation is sufficient to achieve maximum HER-2 protein expression. However, when the incubation time exceeds 40 minutes, the ΔI intensity decreases over time. Therefore, the optimized incubation time is 40 minutes.

[0069] Example 9: Determination of the linear relationship between the peak current intensity of the HER-2 electrochemical sensor and the HER-2 concentration, and drawing a standard curve

[0070] HER-2 standard solutions of different concentrations (0.1-200 ng / mL) were prepared and the peak current intensity was measured using a biosensor. Each concentration was parallel controlled three times. The specific steps were as follows: 5 μL of HER-2 protein antigen of different concentrations was added dropwise to the sensor surface prepared in Example 4, incubated at 37°C for 40 minutes under the optimal incubation conditions, and the unbound antigen was washed with PBS solution. The peak current intensity was detected by differential pulse voltammetry. The linear relationship between the peak current intensity and the HER-2 concentration was obtained through data analysis, which was the standard curve. The results are shown in FIG. Figure 4 The standard relationship curve is I (μA) = -2.191lgCHER-2 +20.552. This indicates a good linear relationship between HER-2 concentration and peak current intensity. As HER-2 concentration increases, the peak current intensity of the electrochemical biosensor increases accordingly. The electrochemical biosensor of the present invention has a low detection limit for HER-2 (0.05 ng / mL), indicating that the sensor has high sensitivity.

Claims

1. A method for preparing methylene blue @ ZnMOF, characterized in that: The bottom-up ultrasonic method is used for preparation, and the preparation steps are as follows: ZnO and 1,3,5-benzenetricarboxylic acid are added to an ethanol aqueous solution at the same time, and then methylene blue is added and stirred, ultrasonically treated to obtain a blue composite material, washed, and dried to obtain methylene blue@ZnMOF crystals.

2. An electrochemical biosensor for detecting HER-2, characterized in that: Its structure is that the surface of the base electrode is covered with a methylene blue@ZnMOF-amino-multi-walled carbon nanotube composite material; a 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide activator is fixed on the surface of the methylene blue@ZnMOF-amino-multi-walled carbon nanotube composite material; a HER-2 antibody is connected to the surface of the activator, and the antibody can specifically recognize the target HER-2 antigen; The methylene blue @ ZnMOF-amino-multi-walled carbon nanotube composite material is prepared by dropwise adding the methylene blue @ ZnMOF and amino-multi-walled carbon nanotubes described in claim 1 onto the surface of a base electrode and drying.

3. A method for preparing an electrochemical biosensor for detecting HER-2, characterized in that: The following steps are involved: (1) Electrode pretreatment: grinding, polishing and ultrasonic cleaning of the base electrode; (2) Synthesis of MB@ZnMOF-AMWCNTs composite material: sequentially adding the aqueous solution of amino-modified multi-walled carbon nanotubes and the aqueous solution of methylene blue@ZnMOF according to claim 1 to the surface of the electrode obtained in step (1), irradiating under an infrared lamp until dry, until the modified material is completely combined with the electrode surface, washing with water, and drying at room temperature; (3) Covalently linking HER2 antibody: 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution and N-hydroxysuccinimide aqueous solution were sequentially added to the electrode obtained in step (2) and allowed to stand at room temperature for 0.5-4 hours; the residual activator was washed with distilled water; then, HER2 antibody Ab solution was added dropwise to the electrode surface and allowed to stand at 0-20°C for 6-24 hours to allow Ab and MB@ZnMO to be fully linked, and then washed with PBS solution and dried at room temperature; (4) Blocking nonspecific sites: Add BSA aqueous solution and let it stand at room temperature for 10-90 minutes to block the unbound sites. Wash with PBS solution and dry at room temperature to obtain an electrochemical biosensor for detecting HER-2.

4. The method for preparing an electrochemical biosensor for detecting HER-2 according to claim 3, wherein: The base electrode is a glassy carbon electrode.

5. The method for preparing an electrochemical biosensor for detecting HER-2 according to claim 3, wherein: In the step (1), the base electrode is polished with Al2O3 powders with particle sizes of 0.1-2 μm and 0.01-0.1 μm, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water in sequence for 1-10 minutes.

6. The method for preparing an electrochemical biosensor for detecting HER-2 according to claim 3, wherein: In step (2), the concentration of the aqueous solution of aminated multi-walled carbon nanotubes is 0.1-8 mg / mL, and the drop volume is 0.5-10 μL; the concentration of the aqueous solution of MB@ZnMOF is 0.1-10 mg / mL, and the drop volume is 0.5-10 μL.

7. The method for preparing an electrochemical biosensor for detecting HER-2 according to claim 3, characterized in that: The concentration of Ab in step (3) is 1-20 μg / mL, preferably, the concentration of Ab is 8-10 μg / mL; more preferably, the concentration of Ab is 8 μg / mL.

8. Use of the electrochemical biosensor according to claim 2 or the electrochemical biosensor prepared by the preparation method according to any one of claims 3 to 7 in the quantitative detection of HER-2, characterized in that: The application method is as follows: after the HER-2 solution to be tested is dripped onto the electrochemical biosensor, the HER-2 protein will be incubated with the antibody pre-modified on the electrode surface to form an antigen-antibody immune complex; by monitoring the current signal of the biosensor, the concentration of the HER-2 solution to be tested is calculated using a standard relationship curve.

9. Use of the electrochemical biosensor according to claim 8 in the quantitative detection of HER-2, characterized in that: The incubation temperature is 10-70°C, and the incubation time is 10-90 minutes; preferably, the incubation temperature is 20-60°C, and the incubation time is 20-60 minutes; more preferably, the incubation temperature is 37°C, and the incubation time is 40 minutes.

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