An electrochemical aptamer sensor for measuring PD-L1 and its preparation method and application

Abstract

The present invention discloses an electrochemical aptamer sensor for measuring PD-L1, and its preparation method and application. The sensor is obtained by specifically binding a MWCNTs-PEI-AuNPs modified electrode connected to a PD-L1 aptamer to PD-L1 and COF-AuNPs-PD-L1 antibody-HRP. The sensor can be further prepared into a kit thereof. The above-mentioned sensor and its kit have high detection sensitivity and strong specificity, and are particularly suitable for the detection of PD-L1 in low-limit samples such as serum and plasma. They are convenient and efficient to use. The preparation steps of the above-mentioned detection sensor include: (1) modification of the substrate electrode; (2) connection of the PD-L1 aptamer; (3) blocking nonspecific sites; (4) specific binding to PD-L1: (5) specific binding to COF-AuNPs-PD-L1 antibody-HRP. The preparation method is green and environmentally friendly and easy to scale up.

Description

Technical Field

[0001] The present invention relates to an electrochemical aptamer sensor for measuring PD-L1, a preparation method and application thereof, and in particular to an electrochemical aptamer sensor for measuring PD-L1 with high detection sensitivity and strong specificity, a preparation method and application thereof. Background Art

[0002] Tumor markers (TM) are substances synthesized and secreted by gene expression in tumor cells during the development and proliferation of malignant tumors, or abnormally produced and / or elevated in response to the body's response to the tumor. These substances can provide effective information for tumor diagnosis, treatment, and prognosis monitoring. New tumor markers offer more possibilities for early diagnosis of cancer, and therefore, the detection and application of new tumor markers hold great promise.

[0003] Programmed death ligand 1 (PD-L1) is expressed by antigen-presenting cells and hematopoietic cells, mediating tumor cell evasion of host immune surveillance. Currently, PD-L1 can be detected in the serum of patients with various cancers and is closely associated with cancer disease progression, survival, and recurrence.

[0004] Currently, the conventional detection method for PD-L1 is ELISA, but the serum PD-L1 content in some patients is below the detection limit and cannot be detected, which has become a technical bottleneck problem that needs to be solved urgently. Summary of the Invention

[0005] Purpose of the invention: In view of the deficiency of existing detection methods in being unable to effectively detect low-level PD-L1, the present invention aims to provide an electrochemical aptamer detection sensor and its kit, preparation method and application that can effectively detect low-level PD-L1 content in serum and plasma.

[0006] Technical solution: As the first aspect of the present invention, the electrochemical aptamer sensor for measuring PD-L1 of the present invention is obtained by specifically binding a MWCNTs-PEI-AuNPs modified electrode connected to a PD-L1 aptamer to PD-L1 and COF-AuNPs-PD-L1 antibody-HRP.

[0007] Aptamers are oligonucleotides (DNA or RNA) that bind to their targets with high affinity and specificity. Compared to antibodies, they offer advantages such as ease of synthesis, low cost, high stability, and ease of modification and storage. Electrochemical aptamer sensors, based on ionic conductivity, utilize the specific binding of aptamers to ligands to capture targets, thereby achieving quantitative detection. Aptamers offer advantages such as high sensitivity, good specificity, and ease of operation.

[0008] The sensor designed in the present invention uses a multi-walled carbon nanotube composite material as the electrode modification material, which can improve the sensitivity of the electrochemical aptamer sensor through its high conductivity; at the same time, COF-AuNPs and HRP enzyme are used to modify the PD-L1 antibody, fully utilizing the properties of COF-AuNPs to increase current and the HRP enzyme cascade reaction to amplify electrical signals, thereby achieving highly sensitive and specific detection performance.

[0009] As a second aspect of the present invention, the method for preparing the detection sensor comprises the following steps:

[0010] (1) Substrate electrode modification: The MWCNTs-PEI-AuNPs solution was placed in contact with the pretreated substrate electrode, and then post-treated;

[0011] (2) Connecting the PD-L1 aptamer: placing the PD-L1 aptamer solution in contact with the electrode prepared in step (1), placing the electrode in contact, and post-processing;

[0012] (3) Blocking nonspecific sites: contacting the electrode prepared in step (2) with the 6-mercapto-1-hexanol solution, allowing it to stand, and post-processing;

[0013] (4) Specific binding to PD-L1:

[0014] (5) Specific binding of COF-AuNPs-PD-L1 antibody-HRP.

[0015] Among them, the base electrode pretreatment method in step (1) is to grind, polish and clean the base electrode. The base electrode is a glassy carbon electrode. The specific method is to polish the base electrode with 0.05 and 0.03 μm Al2O3 powder respectively, and then ultrasonically clean it with anhydrous ethanol and ultrapure water for 5 minutes respectively; the specific method of step (1) is to drop the MWCNTs-PEI-AuNPs solution onto the surface of the pretreated base electrode, place it at 25-45°C for 0.5-3h, preferably at 37°C for 2h, and then wash it with PBS solution and dry it.

[0016] The specific method of step (2) is to drop the PD-L1 aptamer solution onto the surface of the substrate electrode treated in step (1), place it at 1-10°C for 10-15 hours, preferably at 4°C for 12 hours, then wash it with PBS solution and dry it.

[0017] The specific method of step (3) is to drop 6-mercapto-1-hexanol solution onto the surface of the base electrode treated in step (2), place it at room temperature for 0.5 to 5 hours, preferably 2 hours, then wash it with PBS solution and dry it.

[0018] The specific method of step (4) is to add PD-L1 solution to the electrode surface obtained in step (3) for specific reaction, react at 25-45°C for 0.5-3h, preferably at 37°C for 2h, then wash with PBS solution and dry.

[0019] The specific method of step (5) is to add COF-AuNPs-PD-L1 antibody-HRP solution to the electrode surface obtained in step (4) for specific reaction, react at 25-45°C for 0.5-2h, preferably at 37°C for 1h, then wash with PBS solution and dry.

[0020] Specifically, the concentration of the MWCNTs-PEI-AuNPs solution in step (1) is 0.5-5 mg / mL, preferably 1 mg / mL, and the ratio of the dosage volume to the substrate electrode area is 1:5-1:1, preferably 8-15 μL, more preferably 10 μL.

[0021] The concentration of the PD-L1 aptamer solution in step (2) is 0.5 to 5 μM, preferably 1 μM, and the ratio of the dosage volume to the substrate electrode area is 1:5 to 1:1, preferably 8 to 15 μL, and more preferably 10 μL.

[0022] The molar concentration of the 6-mercapto-1-hexanol solution in step (3) is 0.5-5 mM, preferably 1 mM, and the ratio of the dosage volume to the substrate electrode area is 1:5-1:1, preferably 8-15 μL, more preferably 10 μL.

[0023] In step (4), the ratio of the volume of PD-L1 used to the area of ​​the substrate electrode is 1:5 to 1:1, preferably 8 to 15 μL, and more preferably 10 μL.

[0024] In step (5), the concentration of COF-AuNPs-PD-L1 antibody-HRP is 50-500 μg / mL, and the ratio of the dosage volume to the substrate electrode area is 1:5-1:1, preferably 8-15 μL, and more preferably 10 μL.

[0025] More specifically, the method for preparing the MWCNTs-PEI-AuNPs solution used in step (1) comprises the following steps:

[0026] (I) mixing and dispersing MWCNTs and PEI in water; wherein the mass ratio of MWCNTs to PEI is 1:50 to 1:10;

[0027] (II) reacting the HAuCl4 solution with the solution obtained in step (I) and post-treating; wherein the concentration of the HAuCl4 solution is 1 to 20 mg / mL, and the volume ratio of the HAuCl4 solution to the solution obtained in step (I) is 1:10 to 1:1.

[0028] The specific method of step (I) is to dissolve 1-10 mg of MWCNTs and 10-100 mg of PEI in 1-10 mL of water and sonicate for 20-40 minutes, preferably 30 minutes. Furthermore, the MWCNTs are preferably 2 mg, the PEI is preferably 50 mg, and the water is preferably 8 mL.

[0029] The specific method of step (II) is to add a HAuCl4 solution to the solution obtained in step (I), react at 50-100°C for 0.5-3 hours, preferably at 70°C for 2 hours, centrifuge, wash the precipitate with water, resuspend it in water, and ultrasonically disperse it to obtain a MWCNTs-PEI-AuNPs solution. Furthermore, the concentration of the HAuCl4 solution is 1-20 mg / mL, preferably 10 mg / mL, and the volume ratio of the HAuCl4 solution to the solution in step (I) is 1:10-1:1, preferably 1:4; and the washing is performed by immersing the solution in double distilled water for 2-5 times, preferably 3 times.

[0030] More specifically, the preparation method of the COF-AuNPs-PD-L1 antibody-HRP solution used in step (5) comprises the following steps:

[0031] (A) Preparation of COF: PTA and TAB were mixed in DMSO and dispersed; acetic acid was added, dispersed, reacted, and post-treated;

[0032] (B) Preparation of COF-AuNPs: dissolving the COF prepared in step (A), adding sodium citrate solution, heating, then adding HAuCl4 solution, heating, and post-treatment;

[0033] (C) Preparation of COF-AuNPs-PD-L1 antibody-HRP: The COF-AuNPs prepared in step (B) were reacted with the PD-L1 antibody, HRP was added for reaction, and then BSA was added for reaction, and post-processed.

[0034] The specific method of step (A) is to add 20-80 mg of PTA and 50-200 mg of TAB to 20-80 mL of DMSO solution, sonicate for 2-20 minutes, preferably 5 minutes, then add acetic acid, sonicate for 2-20 minutes, preferably 10 minutes, react at room temperature for 10-60 minutes, preferably 30 minutes, and after the reaction, centrifuge to obtain a precipitate, wash the precipitate with ethanol, and vacuum dry to obtain COF. Furthermore, PTA is preferably 50 mg, TAB is preferably 95 mg, and DMSO is preferably 50 mL; ethanol washing is performed by immersing the mixture in ethanol for 2-5 times, preferably 3 times.

[0035] The specific method of step (B) is to add 10 to 50 mL of water, preferably 20 mL of water, to the COF prepared in step (A), and ultrasonicate for 2 to 20 minutes, preferably 10 minutes; then add sodium citrate solution, stir and heat to boiling; then add HAuCl4 solution, stir and heat for 5 to 20 minutes, preferably 15 minutes, centrifuge to obtain a precipitate, wash the precipitate with water, and resuspend it in PBS to obtain a COF-AuNPs solution.

[0036] The specific method of step (C) is to take the COF-AuNPs prepared in step (B), add PD-L1 antibody, react at 1-10°C for 5-15h, preferably at 4°C for 12h; add HRP, react at 1-10°C for 5-15h, preferably at 4°C for 12h; add BSA, react at 1-10°C for 0.5-5h, preferably at 4°C for 1h; centrifuge to obtain a precipitate, wash the precipitate with PBS, and resuspend it in PBS to obtain a COF-AuNPs-PD-L1 antibody-HRP solution.

[0037] Preferably, in the above step (A), the mass ratio of PTA to TAB is 1:10 to 1:1, and the volume ratio of DMSO to acetic acid is 20:1 to 50:1, preferably 25:1.

[0038] In step (B), COF is dissolved to prepare a 0.5-5 mg / mL aqueous solution, preferably a 1 mg / mL aqueous solution; the sodium citrate solution is a 10-50 mM aqueous solution, preferably a 40 mM aqueous solution; the volume ratio of the sodium citrate solution to the COF solution is 1:10-1:5, preferably 1:8; the HAuCl4 solution is a 0.5%-5% by mass aqueous solution, preferably a 1% by mass aqueous solution; the volume ratio of the HAuCl4 solution to the sodium citrate solution is 1:10-1:1, preferably 1:5. Furthermore, the water washing is performed by immersing the solution in double distilled water for 2-5 times, preferably 3 times.

[0039] In step (C), the concentration of COF-AuNPs is 0.5-5 mg / mL, preferably 1 mg / mL; the concentration of PD-L1 antibody is 0.5-5 mg / mL, preferably 1 mg / mL; the volume ratio of COF-AuNPs to PD-L1 antibody is 50:1-500:1, preferably 100:1; the concentration of HRP is 0.5-5 mg / mL, preferably 1 mg / mL; the volume ratio of HRP to PD-L1 antibody is 50:1-500:1, preferably 100:1; and the mass percentage of BSA is 0.2%-2%, preferably 1%. Furthermore, the PBS wash is performed by immersion in PBS for 2-5 times, preferably 3 times.

[0040] As a third aspect of the present invention, the above-mentioned detection sensor constitutes a reagent kit.

[0041] As a fourth aspect of the present invention, the above-mentioned detection sensor and the kit composed thereof can be prepared as a tumor detection product.

[0042] Taking the tumor-related marker PD-L1 as the target, the specific operation steps are as follows: after centrifuging 3 mL of whole blood, take 50 to 500 μL, preferably 100 μL, of the supernatant, and construct an electrochemical aptamer sensor for detection according to the above method.

[0043] The abbreviations of the technical terms in the present invention are as follows:

[0044] MWCNTs: multi-walled carbon nanotubes; PEI: polyethyleneimine; AuNPs: gold nanoparticles; HAuCl4: chloroauric acid; COF: covalent organic framework; PTA: terephthalaldehyde; TAB: 1,3,5-tris(4-aminophenyl)benzene; HRP: horseradish peroxidase; DMSO: dimethyl sulfoxide; BSA: bovine serum albumin; DPV: differential pulse voltammetry.

[0045] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0046] 1. The sensor has high detection sensitivity, reaching the picogram concentration level, and is particularly suitable for the detection of PD-L1 in low-limit samples such as serum and plasma. It also has strong detection specificity, excellent anti-interference performance, and high selectivity for PD-L1.

[0047] 2. The sensor preparation method is green and environmentally friendly and easy to scale up;

[0048] 3. The sensor and its downstream products are easy and efficient to use, with excellent application performance, and have expanded a new model for PD-L1 detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Transmission electron microscopy images of MWCNTs-PEI-AuNPs;

[0050] Figure 2 Transmission electron microscope image of COF;

[0051] Figure 3 Transmission electron microscopy image of COF-AuNPs;

[0052] Figure 4 It is a structural diagram of the detection sensor;

[0053] Figure 5 Cyclic voltammogram for detecting the sensor;

[0054] Figure 6 is a linear relationship graph between the current value and the logarithm of the target concentration;

[0055] Figure 7 This is a diagram to investigate the selectivity of the sensor;

[0056] Figure 8 Figure 2 shows the PD-L1 content in the peripheral blood of healthy people and cancer patients. DETAILED DESCRIPTION

[0057] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0058] Example 1: Preparation of MWCNTs-PEI-AuNPs

[0059] (1) Dissolve 2 mg of MWCNTs and 50 mg of PEI in 8 mL of water and sonicate for 30 min.

[0060] (2) 2 mL of 10 mg / mL HAuCl4 solution was added to the solution obtained in step (1), reacted at 70°C for 2 h, centrifuged, and the precipitate was washed three times with water, resuspended in water, and ultrasonically dispersed to obtain a MWCNTs-PEI-AuNPs solution, the transmission electron microscopy image of which is shown in FIG. Figure 1 shown.

[0061] Example 2: Preparation of COF-AuNPs-PD-L1 Antibody-HRP

[0062] (1) Preparation of COF: 50 mg of PTA and 95 mg of TAB were added to 50 mL of DMSO solution and ultrasonicated for 5 min. Then 2 mL of acetic acid was added and ultrasonicated for 10 min. The mixture was reacted at room temperature for 30 min. After the reaction, the mixture was centrifuged to obtain a precipitate. The precipitate was washed with ethanol three times and vacuum dried to obtain COF. The transmission electron microscopy image is shown in FIG. Figure 2 shown.

[0063] (2) Preparation of COF-AuNPs: 20 mg of COF was added to 20 mL of water and ultrasonicated for 10 min. 2.5 mL of 40 mM sodium citrate solution was added, stirred and heated to boiling. 0.5 mL of 1% HAuCl4 solution was added, stirred and heated for 15 min, and centrifuged to obtain a precipitate. The precipitate was washed with water three times and resuspended in PBS to obtain a COF-AuNPs solution. The transmission electron microscopy image is shown in FIG. Figure 3 shown.

[0064] (3) Preparation of COF-AuNPs-PD-L1 antibody-HRP: Take 1 mL of 1 mg / mL COF-AuNPs, add 10 μL of 1 mg / mL PD-L1 antibody, and react at 4°C for 12 h; add 1 mL of 1 mg / mL HRP, and react at 4°C for 12 h; add 1% by mass BSA, and react at 4°C for 1 h; centrifuge to obtain a precipitate, wash the precipitate three times with PBS, and resuspend it in PBS to obtain a COF-AuNPs-PD-L1 antibody-HRP solution.

[0065] Example 3: Preparation and detection methods of electrochemical aptamer sensors for measuring PD-L1

[0066] The structure of electrochemical aptasensor is as follows Figure 4 As shown, the following steps are included:

[0067] (1) Electrode pretreatment: The glassy carbon electrode was treated with 0.05 and 0.03 μm Al2O3 powder, respectively, and then ultrasonically cleaned with anhydrous ethanol and ultrapure water for 5 min, respectively.

[0068] (2) Adding MWCNTs-PEI-AuNPs: Use a pipette to take 10 μL of 1 mg / mL MWCNTs-PEI-AuNPs solution and drop it on the surface of the pretreated glassy carbon electrode. Place it at 37°C for 2 h to dry. Wash the unbound composite material with PBS solution and let it dry. Take advantage of its larger specific surface area to allow the aptamer to be more enriched on the electrode surface.

[0069] (3) Connecting PD-L1 aptamer: 10 μL of 1 μM PD-L1 aptamer was added to the surface of the glassy carbon electrode obtained in step (2) and placed at 4°C for 12 h. The PD-L1 aptamer was fixed to the electrode surface using gold-sulfur bonds. The unbound aptamer was washed with PBS solution and dried.

[0070] The aptamer sequence for PD-L1 is as follows:

[0071] 5-SH-(CH2)6-GCT-GTG-TGA-CTC-CTG-CAA-GAC-GGA-CCA-GCC-TTG-CCG-CAA-GAC-GGA-CCA-GGG-ATT-CAA-ACG-AGC-AGC-TGT-ATC-TTG-TCT-CC-3 (Reference: Yazdian-RobatiR, et al. An aptamer for recognizing the transmembrane protein PDL-1 (programmed death-ligand 1), and its application to fluorometric single cell detection of human ovarian carcinoma cells. Microchim Acta, 2017(184):4029-4035.).

[0072] (4) Blocking nonspecific sites: Add 10 μL of 1 mM 6-mercapto-1-hexanol solution to the electrode surface obtained in step (3) and leave it at room temperature for 2 h to block the sites not bound by the antibody, prevent certain substances in the sample to be tested from nonspecifically binding to it, and reduce the background signal. Then, wash the unbound 6-mercapto-1-hexanol with PBS solution and dry it.

[0073] (5) Binding to PD-L1: 10 μL of PD-L1 solution was added to the electrode surface obtained in step (4) for specific reaction, and the reaction was carried out at 37°C for 2 h, and then washed with PBS solution and dried.

[0074] (6) COF-AuNPs-PD-L1 antibody-HRP specific binding: 10 μL of 100 μg / mL COF-AuNPs-PD-L1 antibody-HRP solution was added to the electrode surface obtained in step (5) for specific reaction, and the reaction was carried out at 37°C for 1 h, then washed with PBS solution and dried.

[0075] (7) Signal detection: The electrode obtained in step (6) was placed in a deoxygenated 0.01 M PBS solution containing 3 mM hydroquinone and 2 mM hydrogen peroxide to measure the DPV, record the peak current value I, and analyze the results.

[0076] Example 4: Cyclic voltammogram of electrochemical aptasensor for measuring PD-L1

[0077] In order to explore the signal response of the electrochemical aptamer sensor of PD-L1 at each modification stage, the glassy carbon electrode obtained in each step of Example 3 was placed in a 0.01M PBS solution containing 2mM K3[Fe(CN)6] and subjected to cyclic voltammetry scanning at a rate of 0.1V / s. The results are shown in FIG. Figure 5 As shown in Figure 3 , the MWCNTs-PEI-AuNPs modified glassy carbon electrode surface exhibited an increased peak current signal compared to the bare electrode, as the composite enhanced electron transfer and increased sensor sensitivity. Subsequently, the PD-L1 aptamer, 6-mercapto-1-hexanol, PD-L1, and COF-AuNPs-PD-L1 antibody-HRP modified electrode surface exhibited a gradual decrease in the sensor's response signal, as the protein increased the impedance of the electrode surface, reducing the current.

[0078] Example 5: Determination of the Linear Relationship between the Current Value of the Electrochemical Aptamer Sensor for PD-L1 and the Target Concentration

[0079] The preparation method of the electrochemical aptamer sensor is the same as that of Example 3, except that the concentration of PD-L1 in step (5) is different. PD-L1 standard solutions of different concentrations were prepared, namely 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, and 10 ng / mL, and each concentration was paralleled three times. By analyzing the data, a linear relationship between the current value and the logarithm of the target concentration was obtained, and the results are shown in FIG. Figure 6 As the PD-L1 concentration increases, the current value also increases.

[0080] Example 6: Determination of the Selectivity of Electrochemical Aptasensors for PD-L1

[0081] The effects of PD-L1 structural analogues PD-1 and PD-L2 on PD-L1 detection were investigated. The preparation method of the electrochemical aptamer sensor was the same as that in Example 3, except that different substances were used in step (5). The results are shown in FIG. Figure 7 At the same concentration of 1 ng / mL, the current value of PD-L1 is about 8 times that of PD-1 and PD-L2, and the mixed solution has no effect on the detection of PD-L1, indicating that the electrochemical aptamer sensor of the present invention has high specificity and high selectivity for the determination of PD-L1.

[0082] Example 7: Detection of PD-L1 Levels in Peripheral Blood of Healthy Individuals and Cancer Patients

[0083] Healthy and cancer EDTA peripheral whole blood were obtained from healthy people and cancer patients at Taizhou People's Hospital. 3 mL of whole blood was collected from healthy people and cancer patients, respectively. After centrifugation, 100 μL of the supernatant was collected. The sensor was constructed and tested according to Example 3, except that the PD-L1 solution in step (5) was replaced with the supernatant of healthy / cancer patient peripheral blood.

[0084] Among them, the detection range is 1pg / mL-10ng / mL, and the sensitivity is 0.3594pg / mL.

[0085] The obtained electrical signal was converted into concentration according to the standard curve in Example 5 ( Figure 8 ).from Figure 8 It can be found that there is a significant difference in the PD-L1 content in the peripheral blood of healthy people and cancer patients. The PD-L1 content in the peripheral blood of cancer patients is significantly higher than that of healthy people, indicating that the PD-L1 content in peripheral blood has the potential to be used as a new tumor indicator for tumor detection.

Claims

1. An electrochemical aptamer sensor for measuring PD-L1, characterized in that: The MWCNTs-PEI-AuNPs modified electrode connected with the PD-L1 aptamer was specifically bound to PD-L1 and COF-AuNPs-PD-L1 antibody-HRP; The electrochemical aptamer sensor is prepared by the following method: (1) Substrate electrode modification: The MWCNTs-PEI-AuNPs solution was placed in contact with the pretreated substrate electrode, and then post-treated; (2) Connecting the PD-L1 aptamer: placing the PD-L1 aptamer solution in contact with the electrode prepared in step (1), placing the electrode in contact, and post-processing; (3) Blocking nonspecific sites: contacting the electrode prepared in step (2) with the 6-mercapto-1-hexanol solution, allowing it to stand, and post-processing; (4) Specific binding to PD-L1: (5) Specific binding of COF-AuNPs-PD-L1 antibody-HRP.

2. A method for preparing the sensor according to claim 1, characterized in that: The following steps are involved: (1) Substrate electrode modification: The MWCNTs-PEI-AuNPs solution was placed in contact with the pretreated substrate electrode, and then post-treated; (2) Connecting the PD-L1 aptamer: placing the PD-L1 aptamer solution in contact with the electrode prepared in step (1), placing the electrode in contact, and post-processing; (3) Blocking nonspecific sites: contacting the electrode prepared in step (2) with the 6-mercapto-1-hexanol solution, allowing it to stand, and post-processing; (4) Specific binding to PD-L1: (5) Specific binding of COF-AuNPs-PD-L1 antibody-HRP.

3. The preparation method according to claim 2, characterized in that In the step (1), the concentration of the MWCNTs-PEI-AuNPs solution is 0.5-5 mg / mL, and the ratio of the dosage volume to the substrate electrode area is 1:5-1:1; in the step (2), the concentration of the PD-L1 aptamer solution is 0.5-5 μM, and the ratio of the dosage volume to the substrate electrode area is 1:5-1:1; in the step (3), the molar concentration of the 6-mercapto-1-hexanol solution is 0.5-5 mM, and the ratio of the dosage volume to the substrate electrode area is 1:5-1:1; in the step (4), the ratio of the dosage volume of PD-L1 to the substrate electrode area is 1:5-1:1; in the step (5), the concentration of the COF-AuNPs-PD-L1 antibody-HRP is 50-500 μg / mL, and the ratio of the dosage volume to the substrate electrode area is 1:5-1:

1.

4. The preparation method according to claim 2 or 3, characterized in that The preparation method of the MWCNTs-PEI-AuNPs solution in step (1) comprises the following steps: (I) mixing and dispersing MWCNTs and PEI in water; wherein the mass ratio of MWCNTs to PEI is 1:50 to 1:10; (II) reacting the HAuCl4 solution with the solution obtained in step (I) and post-treating; wherein the concentration of the HAuCl4 solution is 1 to 20 mg / mL, and the volume ratio of the HAuCl4 solution to the solution obtained in step (I) is 1:10 to 1:

1.

5. The preparation method according to claim 2 or 3, characterized in that The preparation method of the COF-AuNPs-PD-L1 antibody-HRP solution in step (5) comprises the following steps: (A) Preparation of COF: PTA and TAB were mixed in DMSO and dispersed; acetic acid was added, dispersed, reacted, and post-treated; (B) Preparation of COF-AuNPs: dissolving the COF prepared in step (A), adding sodium citrate solution, heating, then adding HAuCl4 solution, heating, and post-treatment; (C) Preparation of COF-AuNPs-PD-L1 antibody-HRP: The COF-AuNPs prepared in step (B) were reacted with the PD-L1 antibody, HRP was added for reaction, and then BSA was added for reaction, and post-processed.

6. The preparation method according to claim 5, characterized in that In the step (A), the mass ratio of PTA to TAB is 1:10 to 1:1, and the volume ratio of DMSO to acetic acid is 20:1 to 50:

1.

7. The preparation method according to claim 5, characterized in that In the step (B), COF is dissolved to prepare a 0.5-5 mg / mL aqueous solution, the sodium citrate solution is a 10-50 mM aqueous solution, and the volume ratio of the sodium citrate solution to the COF solution is 1:10-1:5; the HAuCl4 solution is a 0.5%-5% mass percentage aqueous solution, and the volume ratio of the HAuCl4 solution to the sodium citrate solution is 1:10-1:

1.

8. The preparation method according to claim 5, characterized in that In step (C), the concentration of COF-AuNPs is 0.5-5 mg / mL, the concentration of PD-L1 antibody is 0.5-5 mg / mL, and the volume ratio of COF-AuNPs to PD-L1 antibody is 50:1-500:1; the concentration of HRP is 0.5-5 mg / mL, and the volume ratio of HRP to PD-L1 antibody is 50:1-500:1; and the mass percentage of BSA is 0.2%-2%.

9. A kit, characterized in that A detection sensor according to claim 1.

10. Use of the sensor according to claim 1 or the kit according to claim 9 in preparing a tumor detection product.

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