Electrochemical immunosensor for detection of mycobacterium tuberculosis antigen esat-6

Abstract

The application provides an electrochemical immunosensor for detecting mycobacterium tuberculosis antigen ESAT-6. 60 The application prepares a composite material MXene / C 60 NPs / Au@Pt, and a large number of ESAT-6 aptamers Apt2 are loaded through the bonding of metal particles and amino groups, and finally form a MXene / C 60 NPs / Au@Pt / Apt2 signal probe solution, which effectively amplifies the electrochemical signal. The introduction of gold nanoparticle loaded MoS2 (MoS2-AuNPs) can promote electron transfer, and the density of ESAT-6 aptamer Apt1 on the electrode is increased by using the biotin avidin system, and a novel electrochemical aptamer biosensor based on double signal output is successfully constructed for the detection of ESAT-6 in a typical sandwich form, which provides a new diagnosis approach for the early diagnosis of tuberculosis patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanotechnology and electrochemical detection technology, and in particular to an electrochemical aptamer sensor for Mycobacterium tuberculosis antigen ESAT-6. BACKGROUND

[0002] Tuberculosis (TB) is a fatal infectious disease caused by Mycobacterium tuberculosis (MTB) infection. According to the statistics of WHO, in the global range, 1060 million people suffered from tuberculosis in 2021, which is equivalent to 134 cases per 100,000 people. In 2021, the total number of TB deaths worldwide was 1.6 million, which was still rising compared with 1.5 million in 2020. Tuberculosis has caused a huge burden on global public health.

[0003] The high prevalence and mortality of tuberculosis are closely related to the delayed diagnosis and misdiagnosis of tuberculosis-infected patients. At present, sputum smear microscopy and bacterial culture of Mycobacterium tuberculosis are the most commonly used methods for clinical diagnosis of tuberculosis. However, the results of sputum smear microscopy of Mycobacterium tuberculosis are affected by factors such as the nature of the specimen, the number of bacteria in the specimen, and the professional skills of the operator, and the positive rate of diagnosis is not high (20%-70%). The culture of Mycobacterium tuberculosis requires an average of 2-4 weeks, and patients with negative sputum smear may require more than 6 weeks of culture time, which is not conducive to the early detection of pulmonary tuberculosis patients and the early diagnosis of drug-resistant pulmonary tuberculosis patients. In recent years, the rapidly developing molecular biology diagnosis technology, including real-time fluorescence polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and whole-genome sequencing (WGS), has the advantages of shorter detection period, higher specificity and biological safety, but most of them require expensive equipment, complicated sample labeling, and high detection cost. There is an urgent need to find a rapid, sensitive, and low-cost tuberculosis diagnosis method to help early detection and early treatment of tuberculosis-infected populations, which is of great significance for the prevention and control of tuberculosis. SUMMARY

[0004] In order to solve the problems in the prior art, in a first aspect, the present application provides an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis antigen ESAT-6, which has the advantages of high specificity and sensitivity, low cost, and fast detection speed, and can be widely used in microbial and pathogen detection.

[0005] Unless otherwise specified, the parts described in the present application are parts by weight, and the percentages are mass percentages.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is:

[0007] MTB antigens appear first when the body is infected with MTB, and can serve as direct evidence of the existence of tubercle bacillus infection, thus having great value in the early diagnosis of tuberculosis. ESAT-6 is an early secretory protein of Mycobacterium tuberculosis, which is encoded by the ESAT-6 gene in the RD1 (deleted regions 1) region of Mycobacterium tuberculosis, and only exists in Mycobacterium tuberculosis and a few pathogenic Mycobacterium. It is absent in BCG (bacillus Calmette-Guérin) and other non-pathogenic Mycobacterium. This feature makes ESAT-6 the best candidate antigen for distinguishing Mycobacterium tuberculosis from non-Mycobacterium tuberculosis, and can distinguish the BCG vaccination situation that may cause false positive results. Moreover, ESAT-6 appears in the early stage of tuberculosis, which is conducive to the early diagnosis of tuberculosis. Therefore, detecting MTB antigen ESAT-6 based on serum immunology has the advantages of short detection time and high specificity, and can also reflect the degree of MTB infection.

[0008] 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:

[0009] The MoS2-AuNPs solution is dropped onto the surface of a clean glassy carbon electrode, and after drying at room temperature, the avidin solution is dropped onto the surface of the modified electrode, and incubated at 3-5 DEG C for 10-12 h; after incubation, the electrode is washed with DEPC water, and the biotin-modified ESAT-6 aptamer (Apt1) is dropped onto the surface of the electrode, and incubated at 3-5 DEG C for 1-2 h, then washed with ultrapure water, and then 0.1-0.5% BSA is dropped, and incubated at room temperature for 45-60 min; then the electrode after incubation for 45-60 min is washed with DEPC water, and the ESAT-6 antigen is dropped onto the surface of the electrode, and incubated at room temperature for 1-2 h, and the electrode is washed with DEPC water, and the MXene / C 60 NPs / Au@Pt / Apt2 solution is dropped onto the surface of the electrode, and incubated at room temperature for 2-3 h, and then washed with DEPC water to obtain the electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen.

[0010] The preparation method of the MXene / C 60 NPs / Au@Pt / Apt2 signal probe solution is as follows: MXene / C 60The aptamer Apt2 of the amino-modified ESAT-6 is added to the NP / Au@Pt dispersion liquid, and stirring is performed in an ice bath for 12-13 h to form a complex. After centrifugation and washing, the complex is redissolved in a PBS solution to form a MXene / C 60 NPs / Au@Pt / Apt2 signal probe solution;

[0011] The MXene / C 60 The preparation method of the NPs / Au@Pt dispersion liquid is as follows: MXene is dissolved in ultrapure water, and ultrasonic treatment is performed until the MXene is uniformly dispersed. C 60 NPs are added, and stirring is performed at room temperature for 15-16 h. After centrifugation and washing, the precipitate is dispersed in ultrapure water. Au@Pt is added, and stirring is performed at room temperature for 1-2 h. After centrifugation and washing, the precipitate is dispersed in ultrapure water to obtain a MXene / C 60 NPs / Au@Pt dispersion liquid;

[0012] The C 60 The preparation method of the C 60 The toluene solution (1 mg / mL) and ultrapure water (5 mL) are placed in an open beaker, and ultrasonic treatment is performed at room temperature until the toluene is completely evaporated. The C 60 The C

[0013] The preparation method of the Au@Pt is as follows: AuNPs are prepared by reducing HAuCl4 with sodium citrate, then HPtCl4 is added for mixing, and ascorbic acid (1 wt%) is used for treatment. Stirring is performed at room temperature until the color becomes black. The transparent solution obtained by restoring the original volume with ultrapure water is the bimetallic core-shell alloy (Au@Pt).

[0014] The preparation method of the base solution MoS2-AuNPs is as follows: MoS2 is added to ultrapure water, and ultrasonic treatment is performed until the MoS2 is uniformly dispersed. Then, HAuCl4 (1 wt%) solution is added for mixing. After magnetic stirring for 15-20 min, sodium borohydride aqueous solution is added. After magnetic stirring for 45-60 min, the MoS2-AuNPs solution is obtained after centrifugation and washing twice with ultrapure water.

[0015] Further, the preparation method of the base solution MoS2-AuNPs is as follows: 1 mg of MoS2 is added to 1 mL of ultrapure water, and ultrasonic treatment is performed until the MoS2 is uniformly dispersed. Then, 200 μL of HAuCl4 (1 wt%) solution is added for mixing. After magnetic stirring for 15 min, 400 μL of 4 mg / mL sodium borohydride aqueous solution is added dropwise. After magnetic stirring for 45 min, the MoS2-AuNPs solution is obtained after centrifugation and washing twice with ultrapure water.

[0016] Specifically, an electrochemical aptamer sensor for detecting mycobacterium tuberculosis antigen ESAT-6, characterized in that the method for constructing the electrochemical aptamer sensor for detecting mycobacterium tuberculosis antigen ESAT-6 comprises the following steps:

[0017] (1) Preparation of signal probe;

[0018] 1) C 60 Preparation of NPs: C 60 NPs were prepared by solvent exchange method, C 60 Toluene solution (1 mg / mL) and ultrapure water (5 mL) were placed in an open beaker and treated with ultrasonic bath at room temperature until toluene was completely evaporated, then C 60 NPs evenly distributed in ultrapure water were obtained;

[0019] 2) Preparation of Au@Pt: AuNPs were prepared by reducing HAuCl4 with sodium citrate, then HPtCl4 was added and mixed, and ascorbic acid (1 wt%) was used for treatment, and the mixture was stirred at room temperature until the color turned black, and then the transparent solution was obtained by restoring the original volume with ultrapure water, which was the bimetallic core-shell alloy (Au@Pt);

[0020] 3) Preparation of MXene / C 60 NPs: 1 mg of MXene was dissolved in 1 mL of ultrapure water, and 1 mL of C 60 NPs prepared in step 1) was added after ultrasonic dispersion, and the mixture was stirred at room temperature for 16 h, then centrifuged, washed, and the precipitate was dispersed in 1 mL of ultrapure water to obtain MXene / C 60 NPs dispersion;

[0021] 4) Preparation of MXene / C 60 NPs / Au@Pt: 1 mL of MXene / C 60 NPs dispersion prepared in step 3) was ultrasonically dispersed, and then 200 uL of Au@Pt prepared in step 2) was added, and the mixture was stirred at room temperature for 1 h, then centrifuged, washed, and the precipitate was dispersed in 1 mL of ultrapure water to obtain MXene / C 60 NPs / Au@Pt dispersion;

[0022] 5) Preparation of MXene / C 60 NPs / Au@Pt / Apt2: 1 mL of MXene / C 60The NPs / Au@Pt dispersion solution was added with 2 μM of Mycobacterium tuberculosis antigen ESAT-6 aptamer Apt2 200 uL, and stirred in an ice bath for 12 h to make it fully anchored on the composite material surface to form a signal probe; after centrifugation and washing, the composite was redissolved in 1 mL of PBS solution and stored at 4°C for standby;

[0023] (2) Preparation of base material MoS2-AuNPs;

[0024] 1 mg of MoS2 was taken and added to 1 mL of ultrapure water, and after ultrasonic dispersion, 200 μL of HAuCl4 (1 wt%) solution was added and mixed, and 400 μL of 4 mg / mL sodium borohydride aqueous solution was added dropwise, and the mixture was magnetically stirred for 15 min to make it fully mixed, and then 400 μL of 4 mg / mL sodium borohydride aqueous solution was added dropwise, and the mixture was magnetically stirred for 45 min. After reaction, the MoS2-AuNPs solution was obtained by centrifugation and washing twice with ultrapure water;

[0025] (3) Construction of an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis antigen ESAT-6:

[0026] 1) The capture probe Apt1 and the signal probe Apt2, as well as the target protein ESAT-6, were treated with 0.1 M PBS (pH = 7.0) buffer at room temperature and stored for standby;

[0027] 2) The glassy carbon electrode was immersed in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 min and then rinsed with ultrapure water for standby;

[0028] 3) The electrode obtained in step 2) was polished to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powder, respectively, and then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water in sequence, and dried for standby;

[0029] 4) The electrode obtained in step 3) was electrochemically activated in 0.5 M H2SO4, and then rinsed with ultrapure water and dried;

[0030] 5) 10 μL of MoS2-AuNPs solution was added to the surface of the cleaned glassy carbon electrode in step 4), and dried at room temperature;

[0031] 6) 10 μL of avidin (100 μg / mL) was added to the electrode prepared in step 5), and incubated at 4°C overnight for 12 h;

[0032] 7) The electrode obtained in step 6) was rinsed with DEPC water, and then 10 μL of biotin-modified ESAT-6 aptamer (Apt1) was added to the electrode surface, and incubated at 4°C for 2 h; after rinsing with ultrapure water, 10 μL of 0.1% BSA was added, and incubated at room temperature for 45 min;

[0033] 8) After the electrode obtained in step 7) is rinsed with DEPC water, different concentrations of ESAT-6 are added dropwise to the surface of the electrode, and incubated at room temperature for 2h;

[0034] 9) After the electrode obtained in step 8) is rinsed with DEPC water, 10μL of MXene / C 60 NPs / Au@Pt / Apt2 signal probe solution is added dropwise, and incubated at 4℃ for 2h, thereby obtaining an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis antigen ESAT-6.

[0035] In a second aspect, the present application also provides a method for detecting ESAT-6 by using the above-mentioned electrochemical aptamer sensor.

[0036] A method for detecting ESAT-6 by using the above-mentioned electrochemical aptamer sensor, characterized in that it comprises the following steps:

[0037] 1) Different concentrations of early secretory protein (ESAT-6) of Mycobacterium tuberculosis are added dropwise to the electrode of the sensor;

[0038] 2) The electrode is placed in 5mL of detection solution (PBS, pH=7.0), and the i-t curve response value is measured; after the background current is stable (50s), 20μL of 200mM H2O2 is added to the solution, and the electrochemical signal change is recorded; after the current signal is stable again, the i-t curve current change value of different concentrations of ESAT-6 is measured; after the measured electrode is rinsed with DEPC water, 4μL of TOAB is added dropwise to excite the intrinsic redox activity of C 60 NPs, and then placed in 0.1M PBS (pH=7.0) solution for characterization, and the DPV response value of different concentrations of ESAT-6 is measured;

[0039] 3) According to the linear relationship between the current change value obtained in step 2) and the logarithmic value of the concentration of ESAT-6, a working curve is drawn;

[0040] 4) The sample to be detected is detected by using the sensor, and the difference in the current change value obtained is calculated by the working curve prepared in step 3) to obtain the concentration of ESAT-6 in the sample to be detected.

[0041] Compared with the prior art, the preparation method and application of the electrochemical aptamer sensor for detecting ESAT-6 have the following outstanding features:

[0042] The MXene / C 60 NPs / Au@Pt prepared by the present application has a stable structure, and can effectively amplify the detection signal, and the C 60The NPs have excellent redox activity, the core-shell bimetallic Au@Pt shows excellent conductivity and catalytic performance, and a novel electrochemical aptamer biosensor based on double signal output is successfully constructed through an aptamer recognition system for detection of Mycobacterium tuberculosis specific antigen ESAT-6. The MoS2-AuNPs are used as a sensing interface in the application, a large amount of aptamer Apt1 is loaded through a biotin avidin amplification system, and signal amplification is further realized. Through the above-mentioned means, the prepared electrochemical aptamer sensor is successfully used for the super-sensitive detection of ESAT-6 in the electrochemical aptamer sensor. Compared with the traditional ESAT-6 detection method, the application has the advantages of high sensitivity, strong specificity, rapid detection, convenient operation, low price of equipment and material, and no pollution, so that a new detection method is provided for the detection of ESAT-6.

[0043] The application has the following beneficial effects:

[0044] 1) The MXene / C 60 NPs / Au@Pt is used for constructing an immunosensor to detect ESAT-6, and shows strong signal amplification effect. 60 The inherent redox activity of the C 60 NPs and the catalytic performance of the Au@Pt realize high-sensitivity detection of ESAT-6 through double signal output. Compared with a single signal response sensor, the double signal response biosensor has better repeatability, high sensitivity and good stability, and the double response method can be cross-verified to improve the reliability of test data. Moreover, the biosensor prepared in the application also has the advantages of wide linear range, strong specificity, short analysis time, good stability and reproducibility.

[0045] 2) The novel composite MXene / C 60 NPs / Au@Pt prepared in the application is mixed with the aptamer Apt2 and stirred to prepare a tracer label through Au-N bond combination, and the method is simple. 60 The tracer label prepared by the novel composite MXene / C 60 NPs / Au@Pt can be applied to various different biosensors.

[0046] 3) The biosensor in the application uses MoS2-AuNPs as a sensing interface, which can increase the conductivity of the electrode on one hand and use the biotin avidin system to load more capture probes on the other hand, so that a sandwich type sensor is constructed through specific combination of Apt1-Ag-Apt2 and a signal probe.

[0047] 4) The biosensor in the application has high specificity for target recognition, which can improve the selectivity of the sensor, so as to provide a new research direction and analysis method for the detection of trace ESAT-6.

[0048] 5) The materials involved in the present application can be synthesized under laboratory conditions, have simple operation, low price of raw materials, low toxicity, environmental friendliness, and extremely small amount of use each time, thereby reducing experimental cost.

[0049] 6) The whole detection and analysis method of the present application is clear and simple, has high sensitivity, and has rapid signal response. The detection limit can reach the fg level.

[0050] 7) The electrochemical aptamer sensor prepared by the method can provide a new method for the detection of ESAT-6. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 Fig. 4 is a calibration curve of logarithmic values of different concentrations of ESAT-6 and the difference in response current of the sensor DPV (Fig. A) and it (Fig. B). Figure 2 Fig. 5 is the stability detection result of the sensor, wherein Fig. A is the DPV response time diagram of the sensor after 20 days of incubation with 100 fg / mL ESAT-6, and Fig. B is the it response result.

[0052] Figure 3 Fig. 6 is the reproducibility result of the sensor obtained by simultaneously incubating five different glassy carbon electrodes of the same batch and two different glassy carbon electrodes of different batches with 10 pg / mL ESAT-6, and then scanning under the same conditions, wherein Fig. A is the DPV result, and Fig. B is the it result. Figure 4 Fig. 7 is the cyclic voltammetry characterization diagram of different modified electrodes in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution with a voltage range from -0.6 to 0.2 V and a scanning rate of 100 mV / s.

[0053] Figure 5 Fig. 8 is the specificity detection diagram of the ESAT-6 immunosensor, wherein the interferents are 0.9% physiological saline, DNA (10 pM), blank control, glucose (5 mM), MPT64 antigen (100 pg / mL), hemoglobin (100 pg / mL), mixture (100 pg / mL), and ESAT-6 antigen (10 pg / mL); Fig. A is the DPV result, and Fig. B is the it result. DETAILED DESCRIPTION

[0054] Additional aspects and advantages of the present application will be partially given in the following description, partially will become obvious from the following description, or will be understood by the practice of the present application. The present application is further described below in combination with examples, but the present application is not limited by this.

[0055] The main chemical reagents used in the embodiments of the present application are as follows:

[0056] Mycobacterium tuberculosis 6 kDa early secretory antigenic target (ESAT-6) was purchased from Shanghai Jingno Biological Technology Co., Ltd. (China, Shanghai). Chloroplatinic acid (HPtCl4), chloroauric acid (HAuCl4), streptavidin (SA) were purchased from Aladdin Biochem Technology Co., Ltd. (China, Shanghai). Layered Ti3C2T X MXene, Fullerene (C 60 NPs), Molybdenum disulfide (MoS2) were purchased from Xianfeng Nanotechnology Co., Ltd. (China, Nanjing). Tetra-n-octylammonium bromide (TOAB) was purchased from Yuanye Biological Co., Ltd. (China, Shanghai). Bovine serum albumin (BSA) was purchased from Beijing Bailingwei Technology Co., Ltd. (China, Beijing). Ethanol was purchased from Chuantung Chemical Group Co., Ltd. Hydrogen peroxide (30%) was purchased from Chengdu Kelong Chemical Co., Ltd. All DNA sequences in the experiment were synthesized and purified by Shanghai Shengong Co., Ltd., and the specific sequences are shown in Table 1.

[0057] Table 1. Nucleotide synthesis sequence table involved in the present application

[0058]

[0059] Equipment and technical parameters used:

[0060] Instrument: Electrochemical detection used a three-electrode system: modified glassy carbon electrode (4 mm in diameter) as the working electrode, platinum wire as the counter electrode, and saturated calomel electrode (SCE) as the reference electrode. Metrohm Autolab B.V. electrochemical workstation (Switzerland Modular instrument) was used for differential pulse voltammetry (DPV) determination. CHI 660E electrochemical workstation (China Shanghai Chenhua instrument) was used for cyclic voltammetry (CV) determination. CHI 660E electrochemical workstation (China Shanghai Chenhua instrument) was used for chronoamperometry (it) determination. pH meter was used to monitor pH value (MP 230, Mettler-Toledo, Switzerland). Cyclic voltammogram (CV) was obtained by electrochemical three-electrode system in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution with a scan rate of 100 mV / s. Differential pulse voltammogram (DPV) was obtained by electrochemical three-electrode system in 0.1 M PBS (pH = 7.0) with a scan rate of 100 mV / s from -0.4 to 0.3 V. Chronoamperometry (it) was obtained by electrochemical three-electrode system in 5 mL 0.1 M PBS (pH = 7.0) with a scan rate of -0.4 V for 50 s, then 20 μL H2O2 (200 mM) was added and scanned again to 100 s.

[0061] Example 1 was operated according to the following steps:

[0062] (1) Preparation of signal probe;

[0063] 1) C 60 Preparation of NPs: C 60 NPs were prepared by solvent exchange method. C 60 NPs were prepared by solvent exchange method. C 60 NPs were prepared by solvent exchange method. C

[0064] 2) Preparation of Au@Pt: AuNPs were prepared by reduction of HAuCl4 with sodium citrate, then HPtCl4 was added and mixed, and ascorbic acid (1 wt%) was added and stirred at room temperature until the color turned black. The transparent solution obtained by restoring the original volume with ultrapure water was the bimetallic core-shell alloy (Au@Pt);

[0065] 3) Preparation of MXene / C 60 NPs: 1 mg of MXene was dissolved in 1 mL of ultrapure water and ultrasonicated until evenly dispersed. Then 1 mL of C 60 NPs prepared in step 1) was added and stirred at room temperature for 16 h. After centrifugation and washing, the precipitate was dispersed in 1 mL of ultrapure water to obtain a MXene / C 60 NPs dispersion;

[0066] 4) Preparation of MXene / C 60 NPs / Au@Pt: 1 mL of MXene / C 60 NPs dispersion prepared in step 3) was ultrasonicated until evenly dispersed, then 200 uL of Au@Pt prepared in step 2) was added and stirred at room temperature for 1 h. After centrifugation and washing, the precipitate was dispersed in 1 mL of ultrapure water to obtain a MXene / C 60 NPs / Au@Pt dispersion;

[0067] 5) Preparation of MXene / C 60 NPs / Au@Pt / Apt2: 2 uM of Mycobacterium tuberculosis antigen ESAT-6 aptamer Apt2 was added to 1 mL of MXene / C 60 NPs / Au@Pt dispersion prepared in step 4) and stirred in an ice bath for 12 h to allow it to be fully anchored on the surface of the composite material to form a signal probe. After centrifugation and washing, the complex was redissolved in 1 mL of PBS solution and stored at 4°C for standby;

[0068] (2) Preparation of the base material;

[0069] 1) MoS2-AuNPs: Take 1 mg of MoS2 and add it to 1 mL of ultrapure water, ultrasonic until dispersed uniformly, then add 200 μL of HAuCl4 (1 wt%) solution, mix well, and then add 400 μL of 4 mg / mL sodium borohydride aqueous solution, magnetically stir for 45 min, then centrifuge and wash twice with ultrapure water to obtain a uniformly dispersed MoS2-AuNPs solution;

[0070] (3) Construction of an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis antigen ESAT-6:

[0071] 1) Treat the capture probe Apt1 and the signal probe Apt2, as well as the target protein ESAT-6, with 0.1 M PBS (pH = 7.0) buffer at room temperature, and store for later use;

[0072] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 min, then rinse with ultrapure water and dry for later use;

[0073] 3) Polish the electrode obtained in step 2) to a mirror finish with 0.3 μm and 0.05 μm Al2O3 powder, respectively, then ultrasonically treat the electrode with ultrapure water, anhydrous ethanol, and ultrapure water in that order, and dry for later use;

[0074] 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0075] 5) Add 10 μL of MoS2-AuNPs solution to the surface of the cleaned glassy carbon electrode obtained in step 4), and dry at room temperature;

[0076] 6) Add 10 μL of avidin (100 μg / mL) to the electrode prepared in step 5), and incubate overnight at 4°C for 12 h;

[0077] 7) Rinse the electrode obtained in step 6) with DEPC water, then add biotin-modified ESAT-6 aptamer (Apt1) to the surface of the electrode, and incubate at 4°C for 2 h; rinse with ultrapure water, then add 10 μL of 0.1% BSA, and incubate at room temperature for 45 min;

[0078] 8) Rinse the electrode obtained in step 7) with DEPC water, then add different concentrations of ESAT-6 to the surface of the electrode, and incubate at room temperature for 2 h;

[0079] 9) Rinse the electrode obtained in step 8) with DEPC water, then add 10 μL of MXene / C 60The NPs / Au@Pt / Apt2 signal probe solution was incubated at 4℃ for 2h to obtain an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis antigen ESAT-6.

[0080] The electrochemical immunosensor constructed in Example 1 was used to detect ESAT-6 according to the following steps

[0081] I. Drawing a standard curve

[0082] 1) The modified electrode of steps 4) to 9) in step (3) of Example 1 was placed in 5mL detection solution (PBS, pH = 7.0) to measure the i-t curve response value. After the background current was stable (50s), 20μL of 200mM H2O2 was added to the solution, and the electrochemical signal change was recorded. After the current signal was stable again, the i-t curve current change value of different concentrations of ESAT-6 was measured. After the measured electrode was washed clean with DEPC water, 4μL of TOAB was added to excite C 60 The intrinsic redox activity of NPs was characterized by placing it in 0.1M PBS (pH = 7.0) solution to measure the DPV response value of different concentrations of ESAT-6;

[0083] The detection results of the sensor for different concentrations of ESAT-6 are shown in Figure 1 A and Figure 1 B. Figure 1 A is a calibration curve of the logarithmic value of different concentrations of ESAT-6 and the difference in DPV current response of the sensor, Figure 1 B is a calibration curve of the logarithmic value of different concentrations of ESAT-6 and the difference in it current response, and the detection results show that the logarithmic value of ESAT-6 concentration has a good linear relationship with both the DPV current response value and the it current response difference of the sensor in the concentration range of 100fg / mL-50ng / mL, and the linear correlation coefficients are 0.9958 and 0.9961, respectively, and the detection limit is 2.88fg / mL.

[0084] 2) The modified electrode of steps 4) to 9) in step (3) of Example 1 was placed in 5mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for CV. The current response signal was measured, and the results are shown in Figure 4 (a) bare glassy carbon electrode; (b) drop MoS2-AuNPs composite material; (c) streptavidin SA; (d) drop biotin-modified aptamer B-ESAT-6; (e) drop BSA; (f) incubate target ESAT-6 A (as shown in Figure 4 ).

[0085] II. Sensor stability test:

[0086] The sensor prepared in Example 1 was stored at 4 DEG C for 20 days, and it was found that the current after 20 days of storage was 90.53% (DPV) and 91.88% (it) of the initial current (as shown in Figure 2 , indicating that the sensor has good stability.

[0087] III. Sensor reproducibility test:

[0088] The sensors prepared by incubating the same batch of five and different batches of two different glassy carbon electrodes with ESAT-6 (10 pg / mL) of the same concentration were measured (as shown in Figure 3 , and the relative standard deviation (RSD) was 2.63% (DPV) and 5.78% (it), indicating that the sensor has good reproducibility.

[0089] IV. Sensor specificity test:

[0090] In order to study the specificity of the proposed adaptive sensor, the DPV response value and the it response value of different interfering substances, such as physiological saline (0.9%), DNA (10 pM) and glucose (5 mM), MPT64 antigen (100 pg / mL), hemoglobin (100 pg / mL), and mixture (100 pg / mL) in 0.1M PBS (pH = 7.0) solution were measured under the same concentration and conditions. The results show (as shown in Figure 5 , the proposed aptamer sensor based on ESAT-6 has good specificity.

[0091] In summary, the present application constructs a new double-signal electrochemical aptamer sensor for rapid and sensitive detection of Mycobacterium tuberculosis 6-kDa early secreted antigenic target (ESAT-6).

[0092] The present application prepares a composite material MXene / C 60 NPs / Au@Pt with a stable structure, and loads a large number of ESAT-6 aptamer Apt2 through the bonding action of metal particles and amino groups, and finally forms a MXene / C 60 NPs / Au@Pt / Apt2 signal probe solution, which effectively amplifies the electrochemical signal. The present application utilizes C 60The NPs and core-shell bimetallic Au@Pt form double signal response, thereby improving the reliability, sensitivity and specificity of test data. The introduction of gold nanoparticle loaded MoS2 (MoS2-AuNPs) can promote electron transfer, while the biotin avidin system is used to increase the density of ESAT-6 aptamer Apt1 on the electrode, and a novel electrochemical aptamer biosensor based on double signal output is successfully constructed for the detection of ESAT-6 in a typical sandwich form, which provides a new diagnostic approach for the early diagnosis of tuberculosis patients.

Claims

1. An electrochemical aptamer sensor for detection of Mycobacterium tuberculosis ESAT-6 antigen, characterized in that, The construction method of the electrochemical aptamer sensor for detecting the Mycobacterium tuberculosis ESAT-6 antigen is as follows: The MoS2-AuNPs solution is dropped onto the surface of a clean glassy carbon electrode, and after drying at room temperature, the avidin solution is dropped onto the surface of the modified electrode, and incubated at 3-5 ℃ for 10-12 h; after the incubated electrode is washed with DEPC water, the biotin-modified ESAT-6 aptamer (Apt1) is dropped onto the surface of the electrode, and incubated at 3-5 ℃ for 1-2 h, and then washed with ultrapure water, and then 0.1-0.5% BSA is dropped, and incubated at room temperature for 45-60 min; after the electrode incubated for 45-60 min is washed with DEPC water, the ESAT-6 antigen is dropped onto the surface of the electrode, and incubated at room temperature for 1-2 h, and then the electrode is washed with DEPC water, and the MXene / C 60 NPs / Au@Pt / Apt2 signal probe solution is dropped onto the surface of the electrode, and incubated at room temperature for 2-3 h, and then washed with DEPC water to obtain an electrochemical aptamer sensor for detecting Mycobacterium tuberculosis ESAT-6 antigen; The MXene / C 60 The preparation method of the MXene / C 60 The preparation method of the MXene / C 60 The preparation method of the MXene / C The MXene / C 60 The preparation method of the NPs / Au@Pt dispersion liquid is: MXene is dissolved in ultrapure water, ultrasonic dispersion is performed until uniform, C 60 NPs is added, and the mixture is placed on a stirrer for stirring at room temperature for 15-16 h; centrifugal separation and washing are performed, the precipitate is dispersed in ultrapure water, Au@Pt is added, and the mixture is placed on a stirrer for stirring at room temperature for 1-2 h; centrifugal separation and washing are performed again, the precipitate is dispersed in ultrapure water, and the NPs / Au@Pt dispersion liquid is obtained. 60 ​ The C 60 The method for preparing the NPs was as follows: 1 mg / mL of C 60 The toluene solution and 5 mL of ultrapure water were placed in an open beaker and treated with an ultrasonic bath at room temperature until the toluene was completely evaporated, and C 60 The NPs were uniformly distributed in ultrapure water; The preparation method of the Au@Pt is as follows: AuNPs are prepared by reducing HAuCl4 with sodium citrate, then HPtCl4 is added for mixing, and the mixture is treated with 1 wt% ascorbic acid, and then stirred at room temperature on a stirrer until the color becomes black, and the transparent solution obtained by restoring the original volume with ultrapure water is the bimetallic core-shell alloy (Au@Pt); The preparation method of the MoS2-AuNPs solution is as follows: MoS2 is taken and added into ultrapure water, ultrasonic dispersion is performed until uniform, then 1 wt% HAuCl4 solution is added for mixing, magnetic stirring is performed for 15-20 min to fully mix, then sodium borohydride aqueous solution is added, and magnetic stirring is performed for 45-60 min, then the MoS2-AuNPs solution uniformly dispersed is obtained by centrifugation and washing twice with ultrapure water.

2. The electrochemical aptamer sensor of claim 1, wherein: The preparation method of the MoS2-AuNPs solution is as follows: 1 mg of MoS2 is taken and added into 1 mL of ultrapure water, ultrasonic dispersion is performed until uniform, then 1 wt% 200 μL of HAuCl4 solution is added for mixing, magnetic stirring is performed for 15 min to fully mix, then 400 μL of 4 mg / mL sodium borohydride aqueous solution is added dropwise, and magnetic stirring is performed for 45 min, then the MoS2-AuNPs solution uniformly dispersed is obtained by centrifugation and washing twice with ultrapure water.

3. A method for detecting ESAT-6 for non-diagnostic or / and therapeutic purposes using the electrochemical aptamer sensor according to claim 1 or 2, characterized in that, The method comprises the following steps: 1) different concentrations of Mycobacterium tuberculosis early secretory protein (ESAT-6) are added dropwise to the sensor electrode; 2) The electrode was placed in 5 mL of PBS solution with pH = 7.0 for detection, and the i-t curve response value was measured. After the background current was stable, 20 μL of 200 mM H2O2 was added to the solution, and the electrochemical signal change was recorded. After the current signal was stable again, the i-t curve current change value of different concentrations of ESAT-6 was measured. After the measured electrode was washed clean with DEPC water, 4 μL of TOAB was added dropwise to excite C 60 After the intrinsic redox activity of the NPs was characterized, they were placed in 0.1 M PBS solution with pH = 7.0 for detection, and the DPV response value of different concentrations of ESAT-6 was measured. 3) a working curve is drawn according to the linear relationship between the current change value obtained in step 2) and the logarithmic value of the ESAT-6 concentration; 4) the sample to be detected is detected by the sensor, and the ESAT-6 concentration in the sample to be detected is calculated by the working curve prepared in step 3) through the current change difference.

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