Electrochemical aptamer sensor and detection method for detection of Mycobacterium tuberculosis ESAT-6 antigen

Abstract

The present invention provides an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT‑6 antigen. The sandwich electrochemical biosensor uses a new type of MXene@Hemin‑AuNPs nanozyme as a signal amplifier and N‑CNTs‑AuNPs as a sensing interface to detect the Mycobacterium tuberculosis-specific antigen ESAT‑6 in the serum of tuberculosis patients, providing a new method for the diagnosis of tuberculosis.

Description

Technical Field

[0001] The invention belongs to the technical field of nanotechnology and electrochemical aptamer sensors, and relates to an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen and a detection method. Background Art

[0002] The World Health Organization (WHO) 2021 Global Tuberculosis Report shows that tuberculosis (TB) is the second leading cause of death among infectious diseases, posing a serious threat to public health security. Reliable diagnostic tools can help detect and treat tuberculosis infection cases early, thus playing an important role in controlling the burden of tuberculosis. Conventional tuberculosis diagnosis includes acid-fast staining and bacterial culture. Acid-fast staining is often used to quickly screen for active pulmonary tuberculosis, but its low sensitivity limits its development; bacterial culture has the advantages of high sensitivity and specificity, while Mycobacterium tuberculosis (MTB) grows slowly and cannot meet the requirements for rapid tuberculosis diagnosis, often leading to delayed treatment. Tuberculosis serological testing can achieve direct on-site tuberculosis diagnosis without long-term culture or complex nucleic acid amplification. In addition, due to the convenience of sampling, this method is effective for children and adults with difficulty in expectoration. Therefore, the use of Mycobacterium tuberculosis-specific antigens for tuberculosis serological testing is a highly promising solution. However, due to the difficulty in preparing specific antibodies and the susceptibility of antibodies to environmental interference, the application of MTB antigens in tuberculosis diagnosis is not satisfactory. Summary of the invention

[0003] In order to solve the problems in the prior art, the present invention provides an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen.

[0004] Unless otherwise specified, all parts described in the present invention are parts by weight and all percentages described are mass percentages.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] An electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen, characterized in that the construction method of the electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen is:

[0007] The N-CNTs-AuNPs solution is dripped onto the clean electrode surface to obtain a modified electrode, and then dried in air; an avidin solution is dripped onto the modified electrode surface, and incubated at 4-5°C for 10-12h; the incubated electrode is washed with DEPC water, and a biotin-modified ESAT-6 aptamer (Ap2) is dripped onto the electrode surface, and incubated at 4-5°C for 1-2h, and then rinsed with ultrapure water and 0.1% BSA is dripped, and incubated at room temperature for 20-30min; the electrode after incubation for 20-30min is then washed with DEPC water, ESAT-6 antigen is dripped onto the electrode surface, and incubated at room temperature for 2-3h, the electrode is washed with DEPC water, MXene@Hemin-Au-Ap2 solution is dripped onto the electrode surface, and incubated at room temperature for 2-3h, and rinsed with DEPC water to obtain the electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen of the present invention.

[0008] The preparation method of the N-CNTs-AuNPs solution is:

[0009] HAuCl4 (1 wt%) was added to the N-CNTs solution, followed by stirring at room temperature for 15-20 min, and then NaBH4 was added and stirred for 40-50 min. After repeated washing and centrifugation with ultrapure water, precipitated N-CNTs-Au was obtained, and the precipitate was redispersed with ultrapure water to form a N-CNTs-AuNPs solution.

[0010] The preparation method of the MXene@Hemin-Au-Ap2 solution is:

[0011] The thiol-modified ESAT-6 aptamer 2 (Ap2) was mixed with the MXene@Hemin-Au solution and stirred continuously for 10-12 h to obtain MXene@Hemin-Au-Ap2 through the Au-S bond, and then centrifuged, the precipitate was washed with water, and dissolved in ultrapure water to form a MXene@Hemin-Au-Ap2 solution.

[0012] The preparation method of the MXene@Hemin-Au solution is:

[0013] First, hemin functionalized MXene (MH) was prepared by direct mixing. The hemin solution was dropped into the MXene solution, treated with ultrasound for half an hour, and then stirred at room temperature overnight to obtain MXene@Hemin. The obtained MXene@Hemin was then washed and centrifuged, and then redispersed in ultrapure water. Subsequently, HAuCl4 (1wt%) was added to the MXene@Hemin, stirred for 60-80 minutes, and centrifuged to obtain a precipitate. After washing, the precipitate was redispersed in ultrapure water to form a MXene@Hemin-Au solution.

[0014] The present invention constructs a sandwich electrochemical biosensor with a new type of MXene@Hemin-AuNPs nanozyme as a signal amplifier and N-CNTs-AuNPs as a sensing interface to detect ESAT-6, a specific antigen of Mycobacterium tuberculosis in the serum of tuberculosis patients. The present invention uses N-CNTs-AuNPs as a sensing interface to accelerate electron transfer, and anchors a large number of high-affinity ESAT-6 aptamers through biotin-avidin bonds for target recognition. The present invention first uses hemin molecules to modify the surface of layered MXene, and then assembles gold nanoclusters through a self-reduction route to prepare a highly active rice enzyme (MXene@Hemin-AuNPs). Through enzyme kinetic analysis, MXene@Hemin-AuNPs has a lower K compared with MXene@Hemin, whether for H2O2 or TMB. m value and faster V max , thus greatly amplifying the detection signal of the biosensor for ESAT-6 antigen. In summary, the present invention uses N-CNTs-AuNPs as the sensing interface and MXene@Hemin-Au as the signal amplifier to achieve ultrasensitive detection of Mycobacterium tuberculosis-specific antigen ESAT-6, providing a new method for the diagnosis of tuberculosis.

[0015] The electrochemical aptasensor for detecting Mycobacterium tuberculosis ESAT-6 antigen is characterized in that before modifying the electrode with the N-CNTs-AuNPs solution, the glassy carbon electrode is first polished on suede with aluminum oxide polishing powder, and then the electrode is ultrasonically cleaned in the order of ultrapure water, anhydrous ethanol and ultrapure water until it becomes a mirror surface.

[0016] The electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen is characterized by comprising the following steps:

[0017] (1) Dissolve the ESAT-6 recombinant protein in DEPC water and store it in enzyme-free tubes at -80°C; dissolve the thiol-modified capture probe in TES buffer containing TCEP and store it at -20°C for future use;

[0018] (2) polishing the glassy carbon electrode on suede with 0.3 and 0.05 μm alumina polishing powder respectively, ultrasonically cleaning the electrode with ultrapure water, anhydrous ethanol and ultrapure water in sequence until it has a mirror surface, and drying it in air for later use;

[0019] (3) 6 μL N-CNTs-AuNPs (1 mg / mL) was added dropwise onto the clean electrode surface and allowed to dry in air;

[0020] (4) 10 μL of avidin (100 μg / mL) was added dropwise to the modified electrode surface and incubated at 4°C for 12 h;

[0021] (6) After the incubation, the electrode was washed with DEPC water, and then the biotin-modified ESAT-6 aptamer (Ap2) was added to the electrode surface and incubated at 4°C for 2 h. After being rinsed with ultrapure water, 10 μL of 0.1% BSA was added and incubated at room temperature for 30 min.

[0022] (7) After the incubated electrode was washed with DEPC water, 10 μL of ESAT-6 antigen of different concentrations was added to the electrode surface and incubated at room temperature for 2 h;

[0023] (8) After the incubated electrode was washed with DEPC water, 15 μL of MXene@Hemin-Au-Ap2 was added to the electrode surface and incubated at room temperature for 2 h;

[0024] (9) The incubated electrode is cleaned with DEPC water to obtain the electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen of the present invention.

[0025] The preparation steps of the N-CNTs-AuNPs are as follows: 200 μL HAuCl4 (1 wt%) is added to 1 mL N-CNTs (1 mg / mL), and then stirred at room temperature for 15 min; then 400 μL NaBH4 (4 mg / mL) is added dropwise, and stirring is continued for 40 min; after repeated washing and centrifugation with ultrapure water, precipitated N-CNTs-Au is obtained, and it is redispersed in ultrapure water to form a 1 mg / mL N-CNTs-AuNPs solution.

[0026] The preparation steps of the probe (MXene@Hemin-Au-Ap2) are as follows: mixing the thiol-modified aptamer 2 (Ap2) with MXene@Hemin, stirring continuously for 12 hours, obtaining MXene@Hemin-Au-Ap2 through a strong Au-S bond, centrifuging the precipitate, washing it with water, and making up the volume with ultrapure water to obtain a MXene@Hemin-Au-Ap2 solution with a final concentration of 1 mg / mL.

[0027] The preparation steps of the MXene@Hemin-Au are as follows: drop a 1 mg / mL hemin solution into a 1 mg / mL MXene solution, and then treat it with ultrasound for half an hour; then gently stir the mixture at room temperature overnight; then wash and centrifuge the obtained MXene@Hemin, and then redisperse the precipitate in ultrapure water; then add 200 μL HAuCl4 (1 wt%) to 1 mL MXene@Hemin and continue stirring for 60 minutes; after the washing step, redisperse the product in ultrapure water, and the final concentration is about 1 mg / mL.

[0028] The present invention also provides a method for detecting Mycobacterium tuberculosis ESAT-6 using the electrochemical aptamer sensor.

[0029] The method for detecting Mycobacterium tuberculosis ESAT-6 using the above-mentioned electrochemical aptamer sensor is characterized by comprising the following steps: placing the electrochemical aptamer sensor for Mycobacterium tuberculosis ESAT-6 antigen detection in a sodium acetate (NaAc-HAc) solution (0.2M, pH=4) containing 1.3mMTMB and 10mM H2O2 for characterization, and measuring the current change value of ESAT-6 with different concentrations; drawing a standard curve according to the relationship between the logarithmic value of different concentrations and the current change value; detecting the sample to be tested using the electrochemical aptamer sensor for Mycobacterium tuberculosis ESAT-6 antigen detection, and calculating the obtained current value through the prepared working curve to obtain the ESAT-6 concentration of the sample to be tested.

[0030] Compared with the prior art, the preparation method and application of the electrochemical aptamer biosensor for detecting ESAT-6 antigen of the present invention have the following outstanding features:

[0031] (1) The present invention successfully prepared MXene@Hemin-AuNPs-aptamers nanozymes and used them as amplifiers to detect ESAT-6 antigens for the first time, showing excellent signal response;

[0032] (2) In the present invention, GCE / N-CNTs-AuNPs is used as the sensing interface, which, on the one hand, increases the conductivity of the electrode, and on the other hand, can immobilize more capture probes, which synergistically amplify the sensor signal with the nanozyme probe, thereby improving the sensitivity and detection range of the electrochemical biosensor for ESAT-6 antigen detection;

[0033] (3) The experimental materials involved in the present invention are easy to obtain and can be completed under laboratory conditions. The entire detection and analysis steps are relatively clear, and the detection limit can reach the fg level. The electrochemical aptamer biosensor for ESAT-6 antigen detection prepared in the present invention is expected to provide a new approach for the diagnosis of tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the construction and detection principle of the electrochemical aptamer biosensor for ESAT-6 antigen detection of the present invention.

[0035] Figure 2 Figure 1 is the detection result of the sensor of the present invention for ESAT-6 antigen of different concentrations: Figure A is the It curve of the sensor scanning ESAT-6 of different concentrations in a sodium acetate (NaAc-HAc) solution (0.2M, pH=4) containing 1.3mM TMB and 10mM H2O2; Figure B is the calibration curve of the logarithmic value of ESAT-6 antigen of different concentrations and the sensor current response value.

[0036] Figure 3 The specificity and stability test results of the sensor of the present invention are shown in Figure A. Figure A is a specificity diagram of incubating different interfering substances; Figure B is a current change trend of the sensor when placed at 4°C for 16 days.

[0037] Figure 4 The results of the reproducibility test of the sensor of the present invention are as follows: a histogram analysis of inter-batch differences and intra-batch differences. DETAILED DESCRIPTION

[0038] The present invention is described in detail below by specific examples. It is pointed out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above invention content. The raw materials and reagents used in the present invention are all commercially available products.

[0039] The main chemical reagents used in the embodiments of the present invention are as follows:

[0040] Layered Ti3C2T X MXene and nitrogen-doped carbon nanotubes (N-CNT) were obtained from Xianfeng Nanotechnology Co., Ltd. (Nanjing, China). Chloroauric acid (HAuCl4), hemin, streptavidin (SA), and tris(2-carboxyethyl)phosphine (TCEP) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was ordered from Beijing Bailingwei Technology Co., Ltd. (Beijing, China). ESAT-6 antibody was purchased from Shanghai Jingnuo Biotechnology Co., Ltd. (Shanghai, China). All other reagents in this study were analytical grade reagents. All DNA sequences in the experiment were synthesized and purified by Shanghai Shenggong Co., Ltd. The specific sequences are shown in Table 1.

[0041] Table 1. Nucleotide synthesis sequence table involved in the present invention

[0042]

[0043] . Equipment and technical parameters: Cyclic voltammetry (CV) and chronoamperometry (IT) were performed using a three-electrode system in a CHI660E electrochemical workstation (Shanghai Chenhua), which included a platinum wire (counter electrode), a saturated calomel electrode (SCE, reference electrode) and a modified glassy carbon electrode (GCE, working electrode). IT curves were obtained by the three-electrode system in a sodium acetate (NaAc-HAc) solution (0.2 M, pH = 4) containing 1.3 mM TMB and 10 mM H2O2 at a constant voltage of 100 mV.

[0044] Example 1 Construction of electrochemical aptamer sensor for detection of Mycobacterium tuberculosis ESAT-6 antigen

[0045] Follow the steps below (build the schematic as shown Figure 1 shown):

[0046] Step 1: 200 μL HAuCl4 (1 wt%) was added to 1 mL N-CNTs (1 mg / mL), and then gently stirred at room temperature for 15 min. Then, 400 μL NaBH4 (4 mg / mL) was slowly dripped into the above mixed solution and stirred for 40 min. After repeated washing with ultrapure water and centrifugation, N-CNTs-Au was obtained and redispersed in ultrapure water for further use.

[0047] Step 2: Preparation of hemin functionalized MXene (MH) by direct mixing method. A 1 mg / mL hemin solution was dropped into a 1 mg / mL MXene solution and then treated with ultrasound for half an hour. The mixture was then gently stirred at room temperature overnight. After this step, the obtained MXene@Hemin was washed and centrifuged several times and then redispersed in ultrapure water. Subsequently, 200 μL HAuCl4 (1 wt%) was added to 1 mL MXene@Hemin and stirred continuously for 60 minutes. After the washing step, the product was redispersed in ultrapure water with a final concentration of approximately 1 mg / mL.

[0048] Step 3: Mix the thiol-modified ESAT-6 aptamer 2 (Ap2) with MXene@Hemin and stir continuously for 12 hours to obtain MXene@Hemin-Au-Ap2 through the Au-S bond, then centrifuge and wash, make up to volume with ultrapure water, and store in a refrigerator at 4°C for future use.

[0049] Step 4: Dissolve the ESAT-6 recombinant protein in DEPC water and dispense into enzyme-free tubes and store at -80°C for later use.

[0050] Step 5: Polish the glassy carbon electrode with 0.3 and 0.05 μm alumina polishing powders respectively, and then ultrasonically clean the electrode surface with ultrapure water, anhydrous ethanol and ultrapure water in sequence until a mirror glassy carbon electrode is obtained, and dry it in air.

[0051] Step 6: Weigh 10 μL of the N-CNTs-AuNPs (1 mg / mL) aqueous solution prepared in step 1 and drop it onto the clean electrode surface, and dry it in air.

[0052] Step 7: Add 10 μL of avidin (100 μg / mL) to the modified electrode surface and incubate at 4°C for 8 h;

[0053] Step 8: After the incubated electrode is washed with DEPC water, the biotin-modified ESAT-6 aptamer is added to the electrode surface and incubated at 4°C for 2h; after being rinsed with ultrapure water, 10μL 0.1% BSA is added and incubated at room temperature for 30min;

[0054] Step 9: After the incubated electrode is washed with DEPC water, 10 μL of ESAT-6 antigen of different concentrations is added to the electrode surface and incubated at room temperature for 2 h;

[0055] Step 10: After washing the incubated electrode with DEPC water, add 10 μL of probe to the electrode surface, incubate at room temperature for 2 hours, and rinse with DEPC water.

[0056] Example 2 Detection of ESAT-6 using electrochemical aptamer biosensor

[0057] The electrochemical aptamer biosensor constructed in Example 1 was used to detect ESAT-6, and the following steps were followed:

[0058] (1) Drawing of standard curve:

[0059] The electrodes of the biosensor constructed in Example 1 were placed in a sodium acetate (NaAc-HAc) solution (0.2M, pH=4) containing 1.3mM TMB and 10mM H2O2 for characterization, and the current values ​​of ESAT-6 at different concentrations were measured. A standard curve was drawn based on the logarithmic value and current signal of ESAT-6 antigen at different concentrations. The test results showed that the two had a good linear relationship in the concentration range of 100fg / mL to 50ng / mL, with a linear correlation coefficient of 0.9919 and a detection limit of 2.36fg / mL. The results are shown in Figure 2 .

[0060] (2) Sensor-specific detection:

[0061] In order to detect the specificity of the sensor of the present invention, the following substances were tested: target ESAT-6 antigen (100 pg / mL), sodium chloride (NaCl, 1 ng / mL), glutathione (GSH, 1 ng / mL), miRNA (1 ng / mL), cytokeratin 19 fragment (CYFRA 21-1, 1 ng / mL) and the mixture. The current response values ​​of different substances measured under the same conditions in a sodium acetate (NaAc-HAc) solution (0.2 M, pH = 4) containing 1.3 mM TMB and 10 mM H2O2 are shown in Figure 2. Figure 3 (A) The results in the figure show that the biosensor of the present invention has satisfactory specificity. Figure 3 The dashed line in (A) represents the blank current value.

[0062] (3) Sensor stability detection:

[0063] The prepared sensor was stored at 4°C for 16 days. The results showed that the current value after storage was still 86% of the initial current, indicating that the sensor has acceptable stability. Figure 3 (B).

[0064] (4) Sensor reproducibility test:

[0065] Under the same conditions, ESAT-6 (1 ng / mL) was measured using 5 different electrodes of the same batch prepared by the present invention. The results are shown in Figure 4 The relative standard deviation (RSD) of the current response value was 2.68%. Three electrodes from different batches (each electrode was tested 3 times) were tested, and the RSD was 0.91%, indicating that the sensor had small intra-batch and inter-batch differences and good reproducibility.

Claims

1. An electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen, characterized in that: The construction method of the electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen is as follows: The N-CNTs-AuNPs solution was dropped onto the clean electrode surface to obtain a modified electrode, which was then air-dried; an avidin solution was dropped onto the modified electrode surface and incubated at 4-5°C for 10-12 h; the incubated electrode was washed with DEPC water, and the biotin-modified ESAT-6 aptamer Ap1 was dropped onto the electrode surface, incubated at 4-5°C for 1-2 h, then rinsed with ultrapure water, and then 0.1% BSA was dropped, and incubated at room temperature for 20-30 min; the electrode was then washed with DEPC water after incubation for 20-30 min, and ESAT-6 antigen was dropped onto the electrode surface, incubated at room temperature for 2-3 h, the electrode was washed with DEPC water, and MXene@Hemin-Au-Ap2 solution was dropped onto the electrode surface, incubated at room temperature for 2-3 h, and rinsed with DEPC water to obtain an electrochemical aptamer sensor for detecting ESAT-6 antigen of Mycobacterium tuberculosis; The preparation method of the N-CNTs-AuNPs solution is: 1 wt% HAuCl4 was added to the N-CNTs solution, and then stirred at room temperature for 15-20 min, and then NaBH4 was added and stirred for 40-50 min, and then the precipitated N-CNTs-AuNPs were obtained after repeated washing and centrifugation with ultrapure water, and the precipitate was redispersed with ultrapure water to form an N-CNTs-AuNPs solution; The preparation method of the MXene@Hemin-Au-Ap2 solution is as follows: the thiol-modified ESAT-6 aptamer Ap2 is mixed with the MXene@Hemin-Au solution, and the mixture is continuously stirred for 10-12 hours to obtain MXene@Hemin-Au-Ap2 through the Au-S bond, and then centrifuged, the precipitate is washed with water, and the precipitate is dissolved with ultrapure water to form a MXene@Hemin-Au-Ap2 solution; The preparation method of the MXene@Hemin-Au solution is as follows: dripping a hemin solution into a MXene solution, treating it with ultrasound for half an hour, and then stirring it at room temperature overnight to obtain MXene@Hemin, then washing and centrifuging the obtained MXene@Hemin, and then redispersing it in ultrapure water, and then adding 1 wt% HAuCl4 to the MXene@Hemin, stirring it continuously for 60-80 minutes, and centrifuging it to obtain a precipitate, and after washing, the precipitate is redispersed in ultrapure water to form a MXene@Hemin-Au solution.

2. The electrochemical aptasensor according to claim 1, characterized in that: Before modifying the electrode with N-CNTs-AuNPs solution, the glassy carbon electrode was polished on suede with alumina polishing powder, and then the electrode was ultrasonically cleaned in the order of ultrapure water, anhydrous ethanol and ultrapure water until it had a mirror surface.

3. The electrochemical aptasensor according to claim 1, characterized in that The steps include: (1) Dissolve the ESAT-6 recombinant protein in DEPC water and store it in enzyme-free tubes at -80°C; dissolve the thiol-modified capture probe in TES buffer containing TCEP and store it at -20°C for future use; (2) Polish the glassy carbon electrode on suede with 0.3 μm and 0.05 μm aluminum oxide polishing powder respectively, and ultrasonically clean the electrode with ultrapure water, anhydrous ethanol and ultrapure water in sequence until it becomes a mirror surface, and dry it in air for later use; (3) Add 6 μL of 1 mg / mL N-CNTs-AuNPs onto the clean electrode surface and dry in air; (4) Add 10 μL of 100 μg / mL avidin onto the modified electrode surface and incubate at 4 °C for 12 h. (5) After the incubated electrode was washed with DEPC water, the biotin-modified ESAT-6 aptamer Ap1 was added to the electrode surface and incubated at 4 °C for 2 h. After being rinsed with ultrapure water, 10 μL of 0.1% BSA was added and incubated at room temperature for 30 min. (6) After the incubation, the electrode was washed with DEPC water, and 10 μL of ESAT-6 antigen of different concentrations was added to the electrode surface and incubated at room temperature for 2 h. (7) After the incubated electrode was washed with DEPC water, 15 μL of MXene@Hemin-Au-Ap2 was added to the electrode surface and incubated at room temperature for 2 h. (8) The incubated electrode is washed with DEPC water to obtain an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen; The preparation steps of the N-CNTs-AuNPs are as follows: 200 μL of 1 wt% HAuCl4 is added to 1 mL of 1 mg / mL N-CNTs, and then stirred at room temperature for 15 min; then 400 μL of 4 mg / mL NaBH4 is added dropwise, and stirring is continued for 40 min; after repeated washing and centrifugation with ultrapure water, precipitated N-CNTs-AuNPs are obtained, and the precipitated N-CNTs-AuNPs are redispersed in ultrapure water to form a 1 mg / mL N-CNTs-AuNPs solution; The preparation steps of the probe MXene@Hemin-Au-Ap2 are as follows: the thiol-modified aptamer Ap2 is mixed with MXene@Hemin, and the mixture is stirred continuously for 12 hours to obtain MXene@Hemin-Au-Ap2 through a strong Au-S bond, and the precipitate is centrifuged, washed with water, and fixed to volume with ultrapure water to obtain a MXene@Hemin-Au-Ap2 solution; The preparation steps of the MXene@Hemin-Au are as follows: drop a 1 mg / mL hemin solution into a 1 mg / mL MXene solution, and then treat it with ultrasound for half an hour; then gently stir the mixture at room temperature overnight; then wash and centrifuge the obtained MXene@Hemin, and then redisperse the precipitate in ultrapure water; then add 200 μL of 1 wt% HAuCl4 to 1 mL MXene@Hemin and continue stirring for 60 minutes; after the washing step, redisperse the product in ultrapure water with a final concentration of 1 mg / mL.

4. A method for detecting Mycobacterium tuberculosis ESAT-6 using the electrochemical aptasensor according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: placing the electrochemical aptamer sensor for detecting ESAT-6 antigen of Mycobacterium tuberculosis in a NaAc-HAc solution containing 1.3 mM TMB and 10 mM H2O2 for characterization, and measuring the current change values ​​of ESAT-6 with different concentrations; drawing a standard curve according to the relationship between the logarithmic values ​​of different concentrations and the current change values; detecting a sample to be tested by using the electrochemical aptamer sensor for detecting ESAT-6 antigen of Mycobacterium tuberculosis, and calculating the ESAT-6 concentration of the sample to be tested by using the obtained current value through the prepared working curve.

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