A graphene chip for identifying signal amplification and preparation method thereof

Abstract

The invention discloses a graphene chip for amplifying identification signals and a preparation method thereof. The graphene chip preparation method comprises the following steps: S1. preparing a nano-gold particle AuNPs solution; S2. preparing a nano-probe main body AuNPs-HRP-Ab2 solution; S3. treating the graphene chip electrode. The preparation method first prepares the nano-probe main body AuNPs-HRP-Ab2 with the corresponding antibody Ab2 for detecting antigens and nano-gold particles, and then couples the nano-probe main body AuNPs-HRP-Ab2 with the graphene chip, which can form a characteristic structure in which one antibody corresponds to multiple enzymes, thereby increasing the number of reaction binding sites on the graphene chip substrate by several times, thereby improving the detection accuracy and detection efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of immunosensors based on graphene chips, and in particular to a graphene chip for amplifying recognition signals and a preparation method thereof. Background Art

[0002] Tumor marker detection is an indispensable operation in the diagnosis of tumor diseases, which can improve the accuracy of tumor disease diagnosis. As one of the common tumor markers, carcinoembryonic antigen is overexpressed in cancer, so it has very important clinical value in the differential diagnosis of malignant tumors, efficacy evaluation and disease monitoring.

[0003] Electrochemical immunosensor technology has the advantages of portability, fast response, high sensitivity and low cost, so it is most commonly used in the determination of carcinoembryonic antigen. In the detection, the signal amplification strategy of most chemical immunosensors currently relies on horseradish peroxidase (HRP) catalyzed hydrogen peroxide (H 2 O 2 ) reduction reaction. In electrochemical immunosensors, the number of enzyme ligase sites directly affects the reduction reaction, as well as the sensitivity and accuracy of detection. In order to improve the detection sensitivity and accuracy of enzyme electrochemical immunosensors, it is usually necessary to modify the substrate such as a chip. The commonly used enzyme modification methods are very simple. For example, when modifying the surface of the substrate, the enzyme is directly connected to the antibody one-to-one. However, due to the limited size of the substrate, the final modified effective binding sites are relatively few, so it is not easy to detect antigen expression with a small signal.

[0004] Therefore, in order to improve detection accuracy and efficiency, it is necessary to improve the modification method based on graphene chips. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides a method for preparing a graphene chip for identification signal amplification, firstly, the corresponding antibody Ab2 for detecting antigen and nano-gold particles are used to prepare the nano-probe main body AuNPs-HRP-Ab2, and then the nano-probe main body AuNPs-HRP-Ab2 is coupled with the graphene chip to form a characteristic structure in which one antibody corresponds to multiple enzymes, thereby increasing the number of reaction binding sites on the graphene chip substrate by several times, thereby improving the detection accuracy and detection efficiency. The present invention also provides a graphene chip prepared by the preparation method.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing a graphene chip for amplifying recognition signals comprises the following steps:

[0008] S1. Preparation of AuNPs solution;

[0009] S2. Preparation of nanoprobe host AuNPs-HRP-Ab2 solution;

[0010] Take equal amounts of horseradish peroxidase HRP solution, gold nanoparticle AuNPs solution and second antibody Ab2 solution, mix and incubate for 1-3 hours, add bovine serum albumin BSA solution, incubate again to obtain the AuNPs-HRP-Ab2 solution for preparing the nanoprobe main body, and store at 3-5°C for future use;

[0011] S3. Processing graphene chip electrodes;

[0012] After acid washing, the graphene chip electrode is immersed in a mixed solution containing EDC and NHS, a first antibody Ab1 solution, a HER2 solution, and a bovine serum albumin BSA solution in sequence, incubated and then washed to obtain an Ab1-HER2 modified graphene chip electrode;

[0013] The graphene chip electrode modified with Ab1-HER2 was immersed in AuNPs-HRP-Ab2 solution of S2, incubated and then washed to prepare the graphene chip electrode modified with Ab1-HER2-Ab2-HRP-AuNPs.

[0014] In the preparation method, the first antibody Ab1 is a monoclonal antibody, and the second antibody Ab2 is a polyclonal antibody. HER2 is specifically recognized and captured by the first antibody Ab1 monoclonal antibody and the second antibody Ab2 polyclonal antibody, and multiple horseradish peroxidases HRP are fixed to achieve a one-to-many connection between antibodies and enzymes.

[0015] Furthermore, in the step S1 of preparing the AuNPs solution, a sodium citrate reduction method is used to prepare the AuNPs solution.

[0016] Furthermore, in the nano-gold particle solution, the nano-gold particles are monodisperse spherical nano-gold particles with a particle size of 10 nm-2 μm.

[0017] Furthermore, in the step S2 of preparing the AuNPs-HRP-Ab2 solution of the nanoprobe body, the following steps are specifically included:

[0018] S21. Prepared at a concentration of 2×10 -3 ~9×10 -2 mmol / L horseradish peroxidase HRP solution;

[0019] S22. Use phosphate buffer solution (PBS) to dilute the AuNPs solution of S1 to obtain a concentration of 5×10 -4 ~7×10 -3 mmol / L AuNPs solution;

[0020] S23. Prepare a second antibody Ab2 solution, and take equal amounts of horseradish peroxidase HRP solution, gold nanoparticle AuNPs solution and second antibody Ab2 solution, vortex and incubate for 2 hours to obtain a vortex solution;

[0021] S24. Prepare a bovine serum albumin (BSA) solution with a mass concentration of 1-2%, and add it to the mixed solution of S23. After mixing and incubating again, store it at 3-5°C for later use to obtain the nanoprobe main body AuNPs-HRP-Ab2 solution.

[0022] Furthermore, in S24, 0.1-0.2 g of bovine serum albumin (BSA) and 10 mL of phosphate buffer solution (PBS) are mixed to prepare a bovine serum albumin (BSA) solution with a mass concentration of 1-2%.

[0023] Furthermore, in the S3 processing of the graphene chip electrode, the following steps are specifically included:

[0024] S31. Immerse the graphene chip electrode in H 2 SO 4 and H 2 O 2 The mixture is pickled in a mixed solution of , and then taken out and rinsed with deionized water for later use;

[0025] S32. Immerse the graphene chip electrode in a mixed solution containing EDC and NHS for 3-5 hours, remove it and rinse it with PBS;

[0026] S33. Immerse the graphene chip electrode in the first antibody Ab1 solution diluted with PBS and incubate at 3-5°C for 3-5h;

[0027] S34. After the graphene chip electrode was rinsed with PBS, it was immersed in a bovine serum albumin BSA solution with a mass concentration of 1-2%, incubated for 30 minutes, rinsed with PBS again, and stored in PBS at 3-5°C to prepare an Ab1-modified graphene chip electrode;

[0028] S35. The Ab1-modified graphene chip electrode was immersed in a HER2 solution, incubated at 3-5°C for 1-3h, rinsed with PBS, immersed in a bovine serum albumin BSA solution with a mass concentration of 1-2%, incubated for 20-50min, and stored in PBS at 3-5°C to prepare an Ab1-HER2-modified graphene chip electrode;

[0029] S36. Immerse the Ab1-HER2 modified graphene chip electrode in the nanoprobe main body AuNPs-HRP-Ab2 solution, incubate at 3-5°C for 1-3h, and rinse with PBS to prepare the Ab1-HER2-Ab-HRP-AuNPs modified graphene chip electrode.

[0030] Furthermore, in the S3 processing of the graphene chip electrode, before pickling, the following steps are also included:

[0031] S30. Graphene chip electrode pretreatment: The graphene chip electrode is completely immersed in deionized water for ultrasonic cleaning, then degreased with acetone, rinsed with deionized water and anhydrous ethanol, and blown dry for later use.

[0032] Furthermore, in the mixed solution of pickling, H 2 SO 4 and H 2 O 2 The ratio is 3-2:1.

[0033] Further, in said S35, the concentrations of the HER2 solution are 10 -16 mol / L、10 -15 mol / L、10 -14 mol / L、10 -12 mol / L and 10 -10 mol / L.

[0034] Another object of the present invention is to provide a graphene chip, which is prepared by the above-mentioned preparation method.

[0035] Based on the above technical solution, the technical effects achieved by the present invention are:

[0036] (1) The method for preparing a graphene chip for recognition signal amplification provided by the present invention innovatively designs a special nanoprobe and prepares a nanoprobe main body AuNPs-HRP-Ab2 solution, which uses gold nanoparticles AuNPs as a solid phase carrier, specifically recognizes and captures HER2 through the first antibody Ab1 monoclonal antibody and the second antibody Ab2 polyclonal antibody, and fixes multiple horseradish peroxidase HRPs to achieve a one-to-many connection between antibodies and enzymes.

[0037] (2) The method for preparing the graphene chip for amplifying the recognition signal provided by the present invention is to couple the carboxylated graphene chip electrode with the nanoprobe body through EDC and NHS chemical coupling agents; the coupled nanoprobe body AuNPs-HRP-Ab2 can increase the number of effective binding sites by several times. A large number of effective binding sites react to further amplify the detection signal, thereby improving the detection sensitivity, accuracy and detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The present invention is a flow chart of the method for preparing a graphene chip.

[0039] Figure 2 This is an electron micrograph of Ab2, HRP and AuNPs of the present invention before vortexing.

[0040] Figure 3 This is an electron microscope image of Ab2, HRP and AuNPs after vortexing of the present invention.

[0041] Figure 4 CV graphs of the graphene chip electrode of the present invention before and after modification with the nanoprobe body.

[0042] Figure 5 It is the electrochemical impedance spectroscopy (EIS) graph of the graphene chip electrode of the present invention before and after being modified with the nanoprobe body. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively in conjunction with specific embodiments and accompanying drawings. Preferred embodiments of the present invention are provided in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0045] Example 1

[0046] Figure 1 This is a flow chart of the method for preparing a graphene chip for amplifying identification signals of this embodiment, as shown in FIG. Figure 1 As shown, this embodiment provides a method for preparing a graphene chip for amplifying recognition signals, which is described by taking breast cancer antigens and corresponding antibodies as examples. The overall preparation method is divided into three steps, mainly including the following steps:

[0047] S1. The AuNPs solution is prepared by sodium citrate reduction method; wherein the AuNPs solution is monodisperse spherical AuNPs with a particle size of 10 nm-2 μm.

[0048] S2. Prepare the nanoprobe main body AuNPs-HRP-Ab2 solution; specifically:

[0049] S21. Prepared at a concentration of 2×10-3 ~9×10 -2 mmol / L HRP solution;

[0050] S22. Dilute the AuNPs solution in S1 with PBS to obtain a concentration of 5×10 -4 ~7×10 -3 mmol / L AuNPs solution;

[0051] S23. Prepare Ab2 solution, and take equal amounts of HRP solution, AuNPs solution and Ab2 solution, vortex and incubate for 2 h to obtain a vortex solution;

[0052] S24. The mixed solution was added into a bovine serum albumin (BSA) solution having a mass concentration of 1%, mixed and incubated again, and then stored at 4° C. for later use, thereby obtaining a nanoprobe main body AuNPs-HRP-Ab2 solution.

[0053] S3. Processing graphene chip electrodes; specifically:

[0054] S30. Pretreatment of graphene chip electrodes: completely immerse the graphene chip electrodes in deionized water for ultrasonic cleaning, then degrease with acetone, rinse with deionized water and anhydrous ethanol, and blow dry for later use;

[0055] S31. Immerse the pretreated graphene chip electrode in H 2 SO 4 and H 2 O 2 The graphene chip electrode is pickled in a mixed solution with a ratio of 3:1, and then rinsed with deionized water for later use; that is, the graphene chip electrode is provided with -COOH groups by pickling;

[0056] S32. The acid-washed graphene chip electrode was immersed in a mixed solution containing EDC and NHS for 4 h, and then removed and rinsed with PBS;

[0057] S33. Immerse the graphene chip electrode in Ab1 solution diluted with PBS and incubate at 4°C for 4h;

[0058] S34. After rinsing the graphene chip electrode with PBS, immersing it in a BSA solution with a mass concentration of 1%, incubating it for 30 minutes, rinsing it with PBS again, and storing it in PBS at 4°C to prepare an Ab1-modified graphene chip electrode; that is, the acid-washed electrode is hung with Ab1 by the EDC-NHS cross-linking method;

[0059] S35. Within the test adaptability range, the concentrations of 10 -16 mol / L、10 -15 mol / L、10 -14 mol / L、10-12 mol / L and 10 -10 mol / L HER2 solution; taking Ab1-modified graphene chip electrodes, respectively placing them in the above HER2 solution, incubating them at 4°C for 2h, rinsing them with PBS, immersing them in a BSA solution with a mass concentration of 1%, incubating them for 30min, and storing them in PBS at 4°C to prepare Ab1-HER2-modified graphene chip electrodes;

[0060] S36. The Ab1-HER2 modified graphene chip electrode was immersed in the nanoprobe main body AuNPs-HRP-Ab2 solution, incubated at 4°C for 2 hours, and rinsed with PBS to prepare the Ab1-HER2-Ab2-HRP-AuNPs modified graphene chip electrode.

[0061] In the graphene chip electrode, the nanoprobe body AuNPs-HRP-Ab2 is coupled to the graphene chip electrode, and the corresponding antibodies Ab1 and Ab2 are mounted for breast cancer antigens.

[0062] During the preparation process, Ab2 was first wrapped together with HRP and covered on gold nanoparticles AuNPs to prepare the nanoprobe body AuNPs-HRP-Ab2. Then, it was coupled to the graphene chip electrode that had been modified with Ab1-HER2 through the EDC-NHS cross-linking method. Through the combination of the nanoprobe body and the antibody, a special structure in which one antibody corresponds to multiple enzymes can be achieved, thereby increasing the reaction binding sites by more than several times on the graphene chip substrate with limited size.

[0063] in,

[0064] AuNPs is the abbreviation of gold nanoparticles;

[0065] HRP is the abbreviation of horseradish peroxidase. Horseradish peroxidase is a glycoprotein composed of colorless enzyme protein and brown iron porphyrin. It contains 18% sugar. It is connected to antibody molecules to label and color the antibodies.

[0066] PBS is the abbreviation of phosphate buffered saline.

[0067] Ab2 is the abbreviation of the second antibody, polyclonal antibody;

[0068] Ab1 is the abbreviation of the first antibody, which is a monoclonal antibody;

[0069] HER2 is the abbreviation for human epidermal growth factor receptor 2, which is an important indicator used to judge the treatment effect of breast cancer patients.

[0070] BSA is bovine serum albumin, a globulin in bovine serum, containing 607 amino acid residues. It is used as a stabilizer in the storage solution and reaction solution of restriction enzymes or modification enzymes, because some enzymes are unstable or have low activity at low concentrations. After adding BSA, it may play a "protective" or "carrier" role. The activity of many enzymes can be greatly improved after adding BSA.

[0071] EDC is the abbreviation of 1-ethyl-(3-dimethylaminopropyl) carbodiimide, which is often used as a carboxyl activator and a phosphate group activator. EDC is used in peptide synthesis, protein and nucleic acid cross-linking, and preparation of immunoconjugates. EDC is often used in combination with N-hydroxysuccinimide (NHS) or sulfonated-NHS to increase the efficiency of the coupling reaction.

[0072] NHS is short for N-hydroxysuccinimide; NHS is used in bioconjugation, cross-linking, labeling and fixation methods; it can control and modify carbodiimide cross-linking reactions involving carboxylates (—COOH) activated by coupling with primary amines (—NH2).

[0073] The BSA solution with a mass concentration of 1% is prepared by mixing 0.1 g of BSA and 10 mL of PBS.

[0074] Example 2

[0075] This embodiment provides a method for preparing a graphene chip for amplifying recognition signals, which is described by taking breast cancer antigens and corresponding antibodies as examples. The overall preparation method is divided into three steps, mainly including the following steps:

[0076] S1. The AuNPs solution is prepared by sodium citrate reduction method; wherein the AuNPs solution is monodisperse spherical AuNPs with a particle size of 10nm-200nm.

[0077] S2. Prepare the nanoprobe main body AuNPs-HRP-Ab2 solution; specifically:

[0078] S21. Prepared at a concentration of 2×10 -3 ~9×10 -2 mmol / L HRP solution;

[0079] S22. Dilute the AuNPs solution in S1 with PBS to obtain a concentration of 5×10 -4 ~7×10 -3 mmol / L AuNPs solution;

[0080] S23. Prepare Ab2 solution, and take equal amounts of HRP solution, AuNPs solution and Ab2 solution, vortex and incubate for 1 hour to obtain a vortex solution;

[0081] S24. The mixed solution was added into a bovine serum albumin (BSA) solution having a mass concentration of 1%, mixed and incubated again, and then stored at 3° C. for later use, thereby obtaining a nanoprobe main body AuNPs-HRP-Ab2 solution.

[0082] S3. Processing graphene chip electrodes; specifically:

[0083] S30. Graphene chip electrode pretreatment: The graphene chip electrode is completely immersed in deionized water for ultrasonic cleaning, then degreased with acetone, rinsed with deionized water and anhydrous ethanol, and then dried for later use;

[0084] S31. Immerse the pretreated graphene chip electrode in H 2 SO 4 and H 2 O 2 The graphene chip electrode is pickled in a mixed solution with a ratio of 2:1, and then rinsed with deionized water for later use; that is, the graphene chip electrode is provided with -COOH groups by pickling;

[0085] S32. The acid-washed graphene chip electrode was immersed in a mixed solution containing EDC and NHS for 3 h, and then removed and rinsed with PBS;

[0086] S33. Immerse the graphene chip electrode in Ab1 solution diluted with PBS and incubate at 3°C ​​for 3h;

[0087] S34. After rinsing the graphene chip electrode with PBS, immersing it in a BSA solution with a mass concentration of 1%, incubating it for 20 minutes, rinsing it with PBS again, and storing it in PBS at 3°C ​​to prepare an Ab1-modified graphene chip electrode; that is, the acid-washed electrode is hung with Ab1 by the EDC-NHS cross-linking method;

[0088] S35. Within the test adaptability range, the concentrations of 10 -16 mol / L、10 -15 mol / L、10 -14 mol / L、10 -12 mol / L and 10 -10 mol / L HER2 solution; taking Ab1-modified graphene chip electrodes, respectively placing them in the above HER2 solution, incubating at 3°C ​​for 1h, rinsing with PBS, immersing them in a BSA solution with a mass concentration of 1%, incubating for 20min, and storing them in PBS at 3°C ​​to prepare Ab1-HER2-modified graphene chip electrodes;

[0089] S36. The Ab1-HER2 modified graphene chip electrode was immersed in the nanoprobe main body AuNPs-HRP-Ab2 solution, incubated at 3°C ​​for 1 hour, and rinsed with PBS to prepare the Ab1-HER2-Ab2-HRP-AuNPs modified graphene chip electrode.

[0090] In the graphene chip electrode, the nanoprobe body AuNPs-HRP-Ab2 is coupled to the graphene chip electrode, and the corresponding antibodies Ab1 and Ab2 are mounted for breast cancer antigens.

[0091] Example 3

[0092] This embodiment provides a method for preparing a graphene chip for amplifying recognition signals, which is described by taking breast cancer antigens and corresponding antibodies as examples. The overall preparation method is divided into three steps, mainly including the following steps:

[0093] S1. The AuNPs solution is prepared by sodium citrate reduction method; wherein the AuNPs solution is monodisperse spherical AuNPs with a particle size of 100 nm-2 μm.

[0094] S2. Prepare the nanoprobe main body AuNPs-HRP-Ab2 solution; specifically:

[0095] S21. Prepared at a concentration of 2×10 -3 ~9×10 -2 mmol / L HRP solution;

[0096] S22. Dilute the AuNPs solution in S1 with PBS to obtain a concentration of 5×10 -4 ~7×10 -3 mmol / L AuNPs solution;

[0097] S23. Prepare Ab2 solution, and take equal amounts of HRP solution, AuNPs solution and Ab2 solution, vortex and incubate for 3 h to obtain a vortex solution;

[0098] S24. The mixed solution was added into a bovine serum albumin (BSA) solution having a mass concentration of 2%, mixed and incubated again, and then stored at 5° C. for later use, thereby obtaining a nanoprobe main body AuNPs-HRP-Ab2 solution.

[0099] S3. Processing graphene chip electrodes; specifically:

[0100] S30. Pretreatment of graphene chip electrodes: completely immerse the graphene chip electrodes in deionized water for ultrasonic cleaning, then degrease with acetone, rinse with deionized water and anhydrous ethanol, and blow dry for later use;

[0101] S31. Immerse the pretreated graphene chip electrode in H 2 SO 4 and H 2 O 2 The graphene chip electrode is pickled in a mixed solution with a ratio of 3:1, and then rinsed with deionized water for later use; that is, the graphene chip electrode is provided with -COOH groups by pickling;

[0102] S32. The acid-washed graphene chip electrode was immersed in a mixed solution containing EDC and NHS for 5 h, and then removed and rinsed with PBS;

[0103] S33. Immerse the graphene chip electrode in Ab1 solution diluted with PBS and incubate at 5°C for 5h;

[0104] S34. After rinsing the graphene chip electrode with PBS, immersing it in a BSA solution with a mass concentration of 1%, incubating it for 50 minutes, rinsing it with PBS again, and storing it in PBS at 5°C to prepare an Ab1-modified graphene chip electrode; that is, the acid-washed electrode is hung with Ab1 by the EDC-NHS cross-linking method;

[0105] S35. Within the test adaptability range, the concentrations of 10 -16 mol / L、10 -15 mol / L、10 -14 mol / L、10 -12 mol / L and 10 -10 mol / L HER2 solution; taking Ab1-modified graphene chip electrodes, respectively entering the above HER2 solution, incubating at 5°C for 3h, rinsing with PBS, immersing in a BSA solution with a mass concentration of 1%, incubating for 50min, and storing in PBS at 5°C to prepare Ab1-HER2-modified graphene chip electrodes;

[0106] S36. The Ab1-HER2 modified graphene chip electrode was immersed in the nanoprobe main body AuNPs-HRP-Ab2 solution, incubated at 5°C for 3 hours, and rinsed with PBS to prepare the Ab1-HER2-Ab2-HRP-AuNPs modified graphene chip electrode.

[0107] In the graphene chip electrode, the nanoprobe body AuNPs-HRP-Ab2 is coupled to the graphene chip electrode, and the corresponding antibodies Ab1 and Ab2 are mounted for breast cancer antigens.

[0108] Example 4

[0109] By adopting the preparation method of Example 1, a graphene chip with amplified identification signals was prepared in this example.

[0110] Figure 2 This is the electron microscope image of Ab2, HRP and AuNPs before mixing in this example. Figure 3 is the electron microscopy image of Ab2, HRP and AuNPs after vortexing in this example, Figure 2 and Figure 3 As shown in Figure 2, in the process of preparing the nanoprobe main body AuNPs-HRP-Ab2 solution, by comparing the electron microscopy images of Ab2 and AuNPs before and after vortexing, it can be seen that Figure 2 In the present invention, the second antibody Ab2 and horseradish peroxidase HRP are discrete proteins, which are not coupled or otherwise connected to the gold nanoparticles AuNPs. Figure 3 In the present invention, gold nanoparticles AuNPs have been coated with proteins (secondary antibody Ab2 and horseradish peroxidase HRP).

[0111] like Figure 3 As shown, the central black dots are AuNPs, and the surrounding light-colored ones are the parts of the nanoprobe second antibody Ab2 and horseradish peroxidase HRP. The nanoprobe second antibody Ab2 and horseradish peroxidase HRP can be well coated on the gold nanoparticles AuNPs, while maintaining a good dispersion state.

[0112] It can be seen that in this embodiment, the nanoprobe body AuNPs-HRP-Ab2 can be successfully prepared by "taking equal amounts of HRP solution, AuNPs solution and Ab2 solution, mixing and incubating for 2 hours to obtain a mixed solution; then adding it to a bovine serum albumin BSA solution with a mass concentration of 1%, mixing and incubating again, and storing it at 4°C for later use".

[0113] Figure 4 CV diagram of the graphene chip electrode of this embodiment before and after modification with the nanoprobe body. Figure 4 As shown, using cyclic voltammetry test, the graphene chip electrode has a significant reduction in the peak values ​​at 0.3V and 0.1V after being modified with the nanoprobe main body AuNPs-HRP-Ab2 solution, indicating that the conductivity of the electrode is reduced after the graphene chip electrode is modified with the nanoprobe main body AuNPs-HRP-Ab2 solution. This is mainly due to the enzymes and antibodies mounted (coupled) to the graphene chip electrode covering the conductive sites on the electrode surface. Due to the large number of mounted enzymes and antibodies, the conductivity of the chip electrode is changed, which also indirectly proves that the reaction signal can be amplified after the graphene chip electrode is modified with the nanoprobe main body AuNPs-HRP-Ab2 solution.

[0114] Figure 5 Graph 1 is the electrochemical impedance spectroscopy (EIS) graph of the graphene chip electrode of this embodiment before and after modification with the nanoprobe body. Figure 5As shown in the figure, after the graphene chip electrode was modified with the nanoprobe main body AuNPs-HRP-Ab2 solution, the electrode impedance changed significantly compared with that before modification. This is mainly due to the large amount of enzymes and antibodies mounted on it changing the electrode impedance. Figure 4 and Figure 5 It can be seen that after the nanoprobe main body AuNPs-HRP-Ab2 is modified to the graphene chip electrode, a large amount of enzymes and antibodies are modified to the surface of the graphene chip electrode, thereby enhancing the detected reaction signal.

[0115] Example 5

[0116] This embodiment provides a method for preparing a graphene chip for amplifying recognition signals. Based on Example 1, the preparation method modifies the corresponding antibodies for other pathogen substances to prepare specific nanoprobe bodies for other antigens, thereby achieving reaction signal amplification of different detection substances.

[0117] The antigen may be carcinoembryonic antigen, or may be a chemical substance such as alpha-fetoprotein or prostate-specific protein that can specifically bind to the capture antibody and the detection antibody.

[0118] For example, anti-acetylcholine receptor detection is required for the diagnosis of myasthenia gravis. Therefore, Ab1 and Ab2 are replaced with anti-acetylcholine receptor antibodies to prepare a specific nanoprobe body for detecting anti-acetylcholine receptors.

[0119] For example, in order to detect colorectal cancer, Ab1 and Ab2 can be replaced with panitumumab (Vectibix), thereby preparing a specific nanoprobe body for detecting colorectal cancer.

[0120] The above contents are merely examples and illustrations of the structure of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and these obvious replacement forms all belong to the protection scope of the present invention.

Claims

1. A method for preparing a graphene chip for amplifying recognition signals, characterized in that: The following steps are involved: S1. Preparation of AuNPs solution; S2. Preparation of nanoprobe main body AuNPs-HRP-Ab2 solution; Take equal amounts of horseradish peroxidase HRP solution, gold nanoparticle AuNPs solution and second antibody Ab2 solution, mix and incubate for 1-3 hours, add bovine serum albumin BSA solution, incubate again to obtain the AuNPs-HRP-Ab2 solution for preparing the nanoprobe main body, and store at 3-5°C for future use; S3. Processing graphene chip electrodes; After acid washing, the graphene chip electrode is immersed in a mixed solution containing EDC and NHS, a first antibody Ab1 solution, a HER2 solution, and a bovine serum albumin BSA solution in sequence, incubated and then washed to obtain an Ab1-HER2 modified graphene chip electrode; The graphene chip electrode modified with Ab1-HER2 was immersed in the AuNPs-HRP-Ab2 solution of S2, incubated and then washed to prepare the graphene chip electrode modified with Ab1-HER2-Ab2-HRP-AuNPs; Wherein, in the preparation of the nanoprobe main body AuNPs-HRP-Ab2 solution in S2, the following steps are specifically included: S21. Prepared at a concentration of 2×10 -3 ~9×10 -2 mmol / L horseradish peroxidase HRP solution; S22. The AuNPs solution of S1 was diluted with phosphate buffer solution (PBS) to obtain a concentration of 5×10 -4 ~7×10 -3 mmol / L AuNPs solution; S23. Prepare a second antibody Ab2 solution, and take an equal amount of horseradish peroxidase HRP solution, gold nanoparticles AuNPs solution and the second antibody Ab2 solution, vortex and incubate for 2h to obtain a vortex solution; S24. Prepare a bovine serum albumin (BSA) solution with a mass concentration of 1-2%, and add it to the mixed solution of S23. After mixing and incubating again, store it at 3-5°C for later use to obtain the nanoprobe main body AuNPs-HRP-Ab2 solution.

2. The method for preparing a graphene chip for amplifying identification signals according to claim 1, characterized in that: In the step S1 of preparing the AuNPs solution, a sodium citrate reduction method is used to prepare the AuNPs solution.

3. The method for preparing a graphene chip for amplifying identification signals according to claim 2, characterized in that: In the nano-gold particle solution, the nano-gold particles are monodispersed spherical nano-gold particles with a particle size of 10 nm-2 μm.

4. The method for preparing a graphene chip for amplifying identification signals according to claim 1, characterized in that: In the S24, 0.1-0.2 g of bovine serum albumin (BSA) and 10 mL of phosphate buffer solution (PBS) are mixed to prepare a bovine serum albumin (BSA) solution with a mass concentration of 1-2%.

5. The method for preparing a graphene chip for amplifying identification signals according to claim 1, characterized in that: In the S3 processing of the graphene chip electrode, the following steps are specifically included: S31. The graphene chip electrode is immersed in a mixed solution of H2SO4 and H2O2 for pickling, and then removed and rinsed with deionized water for later use; S32. The graphene chip electrode was immersed in a mixed solution containing EDC and NHS for 3-5h, and then removed and rinsed with PBS; S33. Immerse the graphene chip electrode in the first antibody Ab1 solution diluted with PBS and incubate at 3-5°C for 3-5h; S34. After rinsing the graphene chip electrode with PBS, immersing it in a bovine serum albumin (BSA) solution with a mass concentration of 1-2%, incubating it for 20-50 minutes, rinsing it with PBS again, and storing it in PBS at 3-5° C. to prepare an Ab1-modified graphene chip electrode; S35. Immerse the Ab1-modified graphene chip electrode in a HER2 solution, incubate at 3-5°C for 1-3h, rinse with PBS, immerse in a bovine serum albumin BSA solution with a mass concentration of 1-2%, incubate for 20-50min, and store in PBS at 3-5°C to prepare an Ab1-HER2-modified graphene chip electrode; S36. The Ab1-HER2 modified graphene chip electrode was immersed in the nanoprobe main body AuNPs-HRP-Ab2 solution, incubated at 3-5°C for 1-3h, and rinsed with PBS to prepare the Ab1-HER2-Ab-HRP-AuNPs modified graphene chip electrode.

6. The method for preparing a graphene chip for amplifying identification signals according to claim 5, characterized in that: In the S3 process of processing the graphene chip electrode, before pickling, the following steps are also included: S30. Graphene chip electrode pretreatment: The graphene chip electrode is completely immersed in deionized water for ultrasonic cleaning, then degreased with acetone, rinsed with deionized water and anhydrous ethanol, and then blown dry for later use.

7. The method for preparing a graphene chip for amplifying identification signals according to claim 5, characterized in that: In the mixed solution of pickling, the ratio of H2SO4 to H2O2 is 3-2:

1.

8. The method for preparing a graphene chip for amplifying identification signals according to claim 5, characterized in that: In S35, the concentrations of the HER2 solutions are 10 -16 mol / L、10 -15 mol / L、10 -14 mol / L、10 -12 mol / L and 10 - 10 mol / L.

9. A graphene chip, characterized in that: The chip is prepared by the method for preparing a graphene chip for amplifying identification signals as described in any one of claims 1 to 8.

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