Nickel-manganese double-metal layered nano enzyme, probe, sensor and construction method and application of nickel-manganese double-metal layered nano enzyme

Abstract

The invention relates to a nickel-manganese double-metal layered (NiMn-LDH) nano enzyme and a preparation method thereof.According to the nickel-manganese double-metal layered (NiMn-LDH) nano enzyme and the preparation method thereof, oxidation-state species (Mn and Ni) with an unstable NiMn-LDH interface serve as driving force of an oxidation-reduction reaction between the oxidation-state species and palladium ions for the first time, no extra reducing agent is needed, safety is good, and the reaction process is mild and controllable; the invention relates to a nickel-manganese bimetal layered nano enzyme probe, which is prepared by combining nickel-manganese bimetal layered nano enzyme NiMn-LDH (at) PdNPs with an amino-modified ESAT-6 aptamer NH2-Apt, and through experiments, the probe greatly enhances the electric signal response; the invention also relates to application of the nickel-manganese bimetallic layered nano-enzyme probe in preparation of a product for detecting a mycobacterium tuberculosis antigen ESAT-6, the product is an electrochemical aptamer biosensor, and experiments prove that the electrochemical aptamer biosensor is high in sensitivity, good in specificity, high in stability, good in repeatability and high in adding standard recovery rate, and the detection limit is 0.629 pg / mL; moreover, experimental raw materials are easy to obtain, steps are clear, and a new way is expected to be provided for diagnosis of tuberculosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanoscale enzyme, electrochemical aptamer biosensor, in particular to a nickel-manganese bimetallic layered nanoscale enzyme, a probe, a sensor, a construction method and an application thereof. BACKGROUND

[0002] Traditional tuberculosis diagnosis requires patients to provide sputum samples, but many patients have dry cough, no sputum or cannot effectively cough out deep sputum and thus are difficult to complete the test. After the body is infected with Mycobacterium tuberculosis (MTB), MTB releases a series of antigens related to active bacterial replication in the body, such as ESAT-6, CFP-10, Ag85 and other MTB specific antigens. These antigens are not affected by the host immune function status, avoiding the existence of intergeneric and interspecific antigenic determinants due to the weak antigenicity of MTB. If these antigens are detected in the serum of patients, they can serve as direct evidence of MTB infection and have high application value in the early screening and diagnosis of tuberculosis. Moreover, the detection sample is the serum of patients, which is easy to obtain. Therefore, MTB specific antigen detection is expected to become a powerful way for tuberculosis screening.

[0003] Currently, the detection methods for MTB specific antigens mainly rely on antigen-antibody immunological assays, such as enzyme-linked immunosorbent assay (ELISA), immunodot method, immunochromatography method, etc. These methods have certain value in clinical application. However, due to the complexity of tuberculosis antigens, it is difficult to obtain specific antibodies with high purity, and the detection is easily disturbed by other high-abundance proteins; the antigen content in the serum of patients in the early stage of the disease is low (below picomolar level), and the sensitivity of existing methods is difficult to achieve accurate determination of MTB antigen targets, which increases the instability, false positives and false negatives of the detection results based on antigen-antibody immunological methods, limiting their application in clinics. Therefore, new breakthroughs are needed for the detection methods based on serum MTB specific antigens.

[0004] A nucleic acid aptamer is a single-stranded nucleotide with high affinity similar to antibodies, which can bind to target ligands with high efficiency and specificity, and has low cost, is easy to modify and has good stability, and has important application value in the treatment and monitoring of diseases. Moreover, the electrochemical aptamer sensing system constructed by combining aptamer recognition technology with electrochemical analysis has high sensitivity, strong specificity and good repeatability, and is a new generation of detection technology that has attracted attention.

[0005] In recent years, with the development of nanotechnology, the application of various new types of nanomaterials provides a new idea for improving the analysis performance of electrochemical aptamer biosensors. In particular, nanoenzymes, a kind of nanomaterial with enzyme-like catalytic characteristics, have unique advantages compared with natural enzymes and traditional enzyme mimics. The combination of nanoenzymes and electroanalysis technology can significantly improve the stability, selectivity, accuracy and electrochemical signal response of electrochemical biosensors, which is conducive to promoting the practical application of electrochemical sensors. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a nickel-manganese bimetallic layered nanometer enzyme, a probe, a sensor, and a construction method and application thereof.

[0007] The technical scheme of the present application is as follows: A preparation method of a nickel-manganese bimetallic layered nanometer enzyme, comprising the following steps: S1. Preparing NiMn-LDH: mixing Ni(NO3)2·6H2O and Mn(CH3COOH)2·4H2O to obtain a mixed solution, adding urotropin to the mixed solution, stirring, and reacting to obtain a precipitate A, which is NiMn-LDH; S2. Preparing a nickel-manganese bimetallic layered nanometer enzyme: dissolving the NiMn-LDH obtained in S1 to obtain a NiMn-LDH solution, dissolving NaPdCl4 to obtain a NaPdCl4 solution, mixing the NiMn-LDH solution and the NaPdCl4 solution, and reacting to obtain a precipitate B, which is a nickel-manganese bimetallic layered nanometer enzyme, denoted as NiMn-LDH@PdNPs.

[0008] Further, the molar ratio of Ni(NO3)2·6H2O: Mn(CH3COOH)2·4H2O: urotropin in step S1 is (0.5-0.8):(0.2-0.4):12.

[0009] Further, the technical parameters for obtaining the precipitate A by adding urotropin to the mixed solution, stirring, and reacting in step S1 are as follows: after adding urotropin to the mixed solution and stirring for 30-60 min, the mixture is transferred into a reaction kettle and reacted at 80-90°C for 10-20 h, and then naturally cooled to room temperature, and the obtained precipitate A is NiMn-LDH.

[0010] Further, step S1 further comprises post-treatment of the precipitate A, and the post-treatment specifically comprises: washing the precipitate A with DMF and anhydrous ethanol multiple times, centrifuging the obtained product, and drying the product at 60°C to obtain NiMn-LDH.

[0011] Further, the mass ratio of NiMn-LDH: NaPdCl4 in step S2 is (1-3): 1.

[0012] The nickel-manganese bimetallic layered nanoscale enzyme is prepared according to the preparation method.

[0013] A nickel-manganese bimetallic layered nanoscale enzyme probe based on a nickel-manganese bimetallic layered nanoscale enzyme, comprising NiMn-LDH@PdNPs and an amino-modified ESAT-6 aptamer NH 2- Apt.

[0014] The preparation method of the nickel-manganese bimetallic layered nanoscale enzyme probe comprises the following steps: dissolving NiMn-LDH@PdNPs to obtain a NiMn-LDH@PdNPs solution, mixing the NiMn-LDH@PdNPs solution with an amino-modified ESAT-6 aptamer solution, stirring in an ice bath for 10-20 h, and then washing, centrifuging and drying the obtained precipitate C to obtain the nickel-manganese bimetallic layered nanoscale enzyme probe, denoted as NiMn-LDH@PdNPs-NH2-Apt.

[0015] Further, the ratio of the NiMn-LDH@PdNPs to the amino-modified ESAT-6 aptamer solution is 1 mg:200 μL.

[0016] The nickel-manganese bimetallic layered nanoscale enzyme probe is used in the preparation of a product for detecting Mycobacterium tuberculosis antigen ESAT-6.

[0017] Further, the product is an electrochemical aptamer biosensor for detecting Mycobacterium tuberculosis antigen ESAT-6. The electrochemical aptamer biosensor comprises a gold nanoparticle modified electrode, streptavidin on the surface of the gold nanoparticle modified electrode, biotin-modified ESAT-6 ligand Bio-Apt, BSA for blocking non-specific sites on the electrode surface, and a nickel-manganese bimetallic layered nanoscale enzyme probe.

[0018] Further, the construction method of the electrochemical aptamer biosensor comprises the following steps: S1. adding streptavidin to the surface of the gold nanoparticle modified electrode, and incubating to obtain a streptavidin modified electrode; S2. loading biotin-modified ESAT-6 ligand Bio-Apt on the surface of the streptavidin modified electrode, and incubating to obtain a Bio-Apt modified electrode; S3. loading BSA on the surface of the Bio-Apt modified electrode to obtain an electrochemical aptamer biosensor substrate; S4. loading ESAT-6 antigen on the surface of the electrochemical aptamer biosensor substrate, incubating, loading the nickel-manganese bimetallic layered nanoscale enzyme probe again, and incubating to obtain an electrochemical aptamer biosensor.

[0019] Further, the construction method of the electrochemical aptamer biosensor, in particular, comprises the following steps: S1. Electrodepositing the pretreated electrode in a 1% HAuCl4 solution to obtain a gold nanoparticle modified electrode, and adding 20 muL of streptavidin with a concentration of 0.1 mg / mL to the surface of the gold nanoparticle modified electrode, and obtaining a streptavidin modified electrode after 4 DEG C incubation; S2. Adding 20 muL of 2 muM biotin modified ESAT-6 ligand Bio-Apt to the interface of the streptavidin modified electrode, and obtaining a Bio-Apt modified electrode after 4 DEG C incubation; S3. Loading 10 muL of 10 mg / mL BSA on the surface of the Bio-Apt modified electrode, reacting for 40-60 min to block the non-specific sites on the electrode surface, and obtaining an electrochemical aptamer biosensor substrate; S4. Washing the surface of the electrochemical aptamer biosensor substrate, incubating after adding ESAT-6 antigen, washing, and adding NiMn-LDH@PdNPs-NH2-Apt to the surface of the electrochemical aptamer biosensor substrate, and obtaining an electrochemical aptamer biosensor after incubation.

[0020] Compared with the prior art, the present application has at least the following advantages: 1. The present application relates to a nickel-manganese bimetallic layered nanometer enzyme and a preparation method thereof, first preparing a nickel-manganese bimetallic layered hydroxide NiMn-LDH, and then preparing a nickel-manganese bimetallic layered nanometer enzyme NiMn-LDH@PdNPs using the NiMn-LDH; the present application first uses the unstable oxidation state species (Mn and Ni) of the NiMn-LDH interface as the driving force for the redox reaction between the palladium ions, without the need for an additional reducing agent, and has the advantages of good safety, a mild and controllable reaction process.

[0021] 2. The present application also relates to a nickel-manganese bimetallic layered nanometer enzyme probe based on the nickel-manganese bimetallic layered nanometer enzyme, which is prepared by combining the nickel-manganese bimetallic layered nanometer enzyme NiMn-LDH@PdNPs with the amino modified ESAT-6 aptamer NH 2- Experiments have proved that the probe greatly enhances the electrical signal response.

[0022] 3. The present application also relates to the application of the nickel-manganese bimetallic layered nanometer enzyme probe in the preparation of a product for detecting the Mycobacterium tuberculosis antigen ESAT-6, and the product is an electrochemical aptamer biosensor for detecting the Mycobacterium tuberculosis antigen ESAT-6; experiments have proved that the electrochemical aptamer biosensor has high sensitivity, good specificity, high stability, good repeatability, high recovery rate, and a detection limit of 0.629 pg / mL; and the experimental raw materials are easy to obtain, the steps are clear, and the electrochemical aptamer biosensor is expected to provide a new way for the diagnosis of tuberculosis. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the fabrication principle of the nickel-manganese layered double hydrogen compound-loaded palladium nanoparticle nanoenzyme probe and electrochemical aptamer sensor of the present invention. Figure 2 These are SEM and TEM images of the nickel-manganese bimetallic layered nanoenzyme NiMn-LDH@PdNPs from Embodiment 3 of the present invention. Figure 3 The It curves and calibration curves for detecting different concentrations of ESAT-6 using the electrochemical aptamer biosensor in Example 4 of this invention are shown. Figure 4 This demonstrates the specific detection capabilities of the electrochemical aptamer biosensor in Embodiment 5 of the present invention. Figure 5 This refers to the stability data of the electrochemical aptamer biosensor of Example 6 of the present invention after 30 days. Figure 6 This is the reproducibility test data for the intra-batch and inter-batch electrochemical aptamer biosensor in Embodiment 7 of the present invention. Detailed Implementation

[0024] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed 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 application content.

[0025] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0026] Source of materials The nucleotide sequence listing involved in this invention is as follows: Example 1: Preparation method of palladium nanozyme probe supported by nickel-manganese layered dihydrogen compounds The schematic diagram of the preparation principle of palladium nanozyme probes supported by nickel-manganese layered dihydrogen compounds is shown below. Figure 1 As shown in A and 1B.

[0027] S1. Preparation of nickel-manganese bimetallic layered nanoenzymes: S11. Preparation of nickel-manganese double-metal layered hydroxide NiMn-LDH: Ni(NO3)2.6H2O (nickel nitrate hexahydrate, 175 mg, 0.6 mmol) and Mn(CH3COOH)2.4H2O (manganese acetate, 49 mg, 0.2 mmol) were dissolved in 20 mL ultrapure water, and then ultrasonically treated at room temperature for 5 min; afterwards, urotropine (1.68 g, 12 mmol) was added to the above solution, stirred for 30 min, and the obtained mixture was transferred to a Teflon high-pressure reaction kettle, and reacted at 80°C for 12 h. After natural cooling to room temperature, the precipitate was washed to neutral, and the green precipitate was washed with DMF (N,N-dimethylformamide) and ethanol for 3 times, respectively, and dried at 60°C after centrifugation at 12000 rpm to obtain NiMn-LDH; S12. Preparation of double-metal layered nanoscale enzyme NiMn-LDH@PdNPs by self-reduction method: 1 mg of NiMn-LDH was added to 1 mL of a water-absolute ethanol mixture (v / v=2:1) and ultrasonically treated for 3 min; under magnetic stirring, 200 μL of a NaPdCl4 solution (1 wt.%) was dropped into the NiMn-LDH solution, and reacted at room temperature for 45 min; the obtained precipitate was centrifuged and washed several times, and then dried at 60°C to obtain NiMn-LDH@PdNPs; S2. Preparation of nanoscale enzyme probe NiMn-LDH@PdNPs-NH2-Apt: 1 mL of NiMn-LDH@PdNPs with a concentration of 1 mg / mL was mixed with 200 μL of an amino-modified ESAT-6 Mycobacterium tuberculosis antigen aptamer (NH2-Apt) solution, and stirred in an ice bath for 12 h. Finally, the obtained precipitate was centrifuged and washed several times, dispersed in 1 mL of ultrapure water, and stored at 4°C for standby use.

[0028] Example Two Preparation method of electrochemical aptamer sensor with nanoscale enzyme catalytic amplification The electrochemical aptamer sensor with nanoscale enzyme catalytic amplification prepared in this example is used for specific detection of Mycobacterium tuberculosis antigen ESAT-6, and the preparation process is as follows, and the preparation principle diagram is as shown in Figure 1 C.

[0029] S1. A glassy carbon electrode was polished on a chamois leather with 300 nm and 50 nm alumina polishing powder, respectively, and the electrode was ultrasonically cleaned in the order of ultrapure water, absolute ethanol, and ultrapure water until it became mirror-like. Then, the electrode was activated in an H2SO4 solution (0.5 M), and a potential scan was performed at a scan rate of 0.1 V / s, ranging from -0.35 V to +1.6 V, until a reproducible cyclic voltammogram was obtained. Then, the electrode was washed with deionized water and dried in air for standby use.

[0030] S2. The prepared electrode was electrodeposited in HAuCl4 (tetrachloroauric acid, 1%) solution for 30 seconds to modify the electrode with nano-gold for further electrode construction.

[0031] S3. 20 μL of streptavidin (SA, 0.1 mg / mL) was dropped on the surface of the above modified electrode and incubated at 4°C for 12 hours.

[0032] S4. 20 μL of biotin-modified ESAT-6 ligand (Bio-Apt, 2 μM) was dropped on the above electrode interface and incubated at 4°C for 2 hours.

[0033] S5. 10 μL of 10 mg / mL BSA was dropped on the above electrode interface and reacted at room temperature for 45 minutes to block the non-specific sites on the electrode surface.

[0034] S6. After the above electrode was washed with ultrapure water, 20 μL of ESAT-6 antigen of different concentrations was dropped on the electrode surface and incubated at room temperature for 2 hours.

[0035] S7. After the above electrode was washed with ultrapure water, 20 μL of the nano-enzyme probe (NiMn-LDH@PdNPs-NH2-Apt) prepared in Example One was dropped on the electrode surface and incubated at room temperature for 2 hours to complete the construction.

[0036] Example Three Morphological characterization of NiMn-LDH@PdNPs nano-enzyme In this example, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the morphology of the nickel-manganese bimetallic layered nano-enzyme NiMn-LDH@PdNPs, Figure 2 A is a SEM image, Figure 2 B is a TEM image, from Figure 2 As can be seen from A, the fine granular change on the multi-layered overlapped structure of NiMn-LDH confirms that PdNPs are loaded on its surface, Figure 2 B The TEM image further confirms the lamellar structure and the loading of metal particles. The black dots in the image represent metal particles.

[0037] Example Four Detection of antigen ESAT-6 using electrochemical aptamer biosensor Chronoamperometry: cyclic voltammetry (CV) and chronoamperometry (it) measurements were performed in a CHI660E electrochemical workstation (Shanghai Chenhua) using a three-electrode system, which included a platinum wire (counter electrode), a saturated calomel electrode (SCE, reference electrode), and a modified glassy carbon electrode (GCE, working electrode). The It curve was obtained by the three-electrode system in 1.3 mM TMB and 10 mM H2O2 in a sodium acetate (NaAc-HAc) solution (0.2 M, pH = 4) at a constant voltage of 100 mV.

[0038] The electrode of the biosensor constructed in Example 2 was characterized by placing it in a mixed solution containing 4 mL of TMB-H202, and the It current response values at different concentrations of ESAT-6 were measured by chronoamperometry. The concentrations of ESAT-6 were 10 ng / mL, 50 ng / mL, 1 ng / mL, 500 pg / mL, 250 pg / mL, 100 pg / mL, 75 pg / mL, and 0, respectively. A standard curve was plotted according to the logarithmic values of different concentrations of ESAT-6 antigens and the current signals. The It curve at different target concentrations is shown in FIG. 2A, and the calibration curve of the logarithmic values of different concentrations of ESAT-6 antigens and the sensor current response values is shown in FIG. 2B. As can be seen from FIG. 2B, the two have a good linear relationship in the concentration range of 10 ng / mL to 75 pg / mL. The linear correlation coefficient R is 0.97813. The detection limit is calculated by the formula LOD = 3σ / S, where σ represents the standard deviation of the blank, and S represents the slope of the calibration curve. The detection limit is 0.629 pg / mL. Figure 3 A. The It curve at different target concentrations is shown in FIG. 2A, and the calibration curve of the logarithmic values of different concentrations of ESAT-6 antigens and the sensor current response values is shown in FIG. 2B. As can be seen from FIG. 2B, the two have a good linear relationship in the concentration range of 10 ng / mL to 75 pg / mL. The linear correlation coefficient R is 0.97813. The detection limit is calculated by the formula LOD = 3σ / S, where σ represents the standard deviation of the blank, and S represents the slope of the calibration curve. The detection limit is 0.629 pg / mL. Figure 3 B. It can be seen from the figure that the current responses of the Blank, DNA, CFP-10, MPT64, and CRP groups are low, and the current response values of the ESAT-6 and Mix groups are high. The current response values of the Mix group and the ESAT-6 group are close, indicating that the biosensor prepared in the application has satisfactory specificity and strong anti-interference ability. Figure 3

[0039] Example Five: Specific detection of the sensor In order to detect the specificity of the sensor of the application, non-tuberculosis proteins, tuberculosis-related proteins, non-protein substances, etc. were detected in this embodiment, including Blank, a random sequence DNA (2 pM, see SEQ ID NO. 3), CFP-10 protein (1 ng mL -1 ), MTB protein 64 (MPT64 antigen, 1 ng mL -1 ), C-reactive protein (CRP, 1 ng mL -1 ), ESAT-6 (1 ng mL -1 ), and a mixture of MPT64 antigen and ESAT-6 (1 ng mL -1 ). The detection results are shown in FIG. 2C. As can be seen from the figure, the current responses of the Blank, DNA, CFP-10, MPT64, and CRP groups are low, and the current response values of the ESAT-6 and Mix groups are high. The current response values of the Mix group and the ESAT-6 group are close, indicating that the biosensor prepared in the application has satisfactory specificity and strong anti-interference ability. Figure 4

[0040] Example Six: Stability detection of the sensor The sensor prepared in Example Two was stored at 4°C for 10, 20, and 30 days, respectively. The test results of the sensor are shown in FIG. 3. It was found that the current value was still about 80% of the initial current after 30 days of storage, indicating that the sensor had acceptable stability. Figure 5 ​​​

[0041] Example Seven Sensor reproducibility detection The same batch of five different electrodes prepared using the preparation method of Example Two of the present application were used to detect the antigen ESAT-6, and the detection results are shown in Figure 6 The relative standard deviation (RSD) of the current response value was 6.74%, and the RSD of three electrodes of different batches was 4.21%, indicating that the batch and batch differences of the sensor were small, and the reproducibility was good.

[0042] Example Eight Sensor standard addition recovery analysis To evaluate the clinical application potential of the sensor constructed by the present application, the recovery rate test (Table 1) was performed on the proposed method using a standard addition serum sample (prepared by adding the target to the serum sample of a normal person diluted 50 times). The recovery rate was 97.8% to 113.1%, and the RSD was <5.5%, indicating that the sensor prepared by the method described in the present application can be used for detecting ESAT-6 antigen in serum samples.

[0043] Table 1 Sensor standard addition recovery detection results prepared by the present application Example Nine Nanoplasma kinetic parameter analysis In this embodiment, the catalytic performance of the designed NiMn-LDH@PdNPs nanoplasma was evaluated by enzyme kinetics: steady-state reaction kinetics analysis was completed by changing the concentration of TMB (under a fixed H2O2 concentration) or vice versa changing the concentration of H2O2, according to the formula 1 / V = K m / V max x 1 / [S] + 1 / V max for calculation, where V represents the initial speed, and [S] represents the substrate concentration. According to the calculation, it has a lower K m value and a faster reaction rate for H2O2 and TMB, indicating that the designed nanoplasma has the potential to drive the electrochemical aptamer sensor to amplify the electrical signal.

[0044] Table 2 Enzyme kinetic parameters of NiMn-LDH@PdNPs nanoplasma The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.

Claims

1. A preparation method of a nickel-manganese bimetallic layered nanoscale enzyme, characterized in that, Comprising the following steps: S1. Preparation of NiMn-LDH: mix Ni(NO3)2·6H2O with Mn(CH3COOH)2·4H2O to obtain a mixed solution, add urotropin to the mixed solution, stir, and react to obtain precipitate A, which is NiMn-LDH; S2. Preparation of nickel-manganese bimetallic layered nanoscale enzyme: dissolve the NiMn-LDH obtained in S1 to obtain a NiMn-LDH solution, dissolve NaPdCl4 to obtain a NaPdCl4 solution, mix the NiMn-LDH solution with the NaPdCl4 solution, and react to obtain precipitate B, which is nickel-manganese bimetallic layered nanoscale enzyme, denoted as NiMn-LDH@PdNPs.

2. The production method according to claim 1, characterized by, The molar ratio of Ni(NO3)2·6H2O: Mn(CH3COOH)2·4H2O: urotropin in step S1 is (0.5-0.8):(0.2-0.4):

12.

3. The production method according to claim 2, characterized by, The mass ratio of NiMn-LDH: NaPdCl4 in step S2 is (1-3):

1.

4. The production method according to claim 3, characterized by, In step S1, the technical parameters for adding urotropin to the mixed solution, stirring, and reacting to obtain precipitate A are as follows: after adding urotropin to the mixed solution and stirring for 30-60 min, transfer the solution to a reaction kettle and react at 80-90°C for 10-20 h, then naturally cool to room temperature, and the obtained precipitate A is NiMn-LDH.

5. The nickel-manganese bimetallic layered nanoscale enzyme prepared by the preparation method of any one of claims 1-4.

6. A nickel-manganese bimetallic layered nanoscale enzyme probe based on nickel-manganese bimetallic layered nanoscale enzyme, characterized by, comprising NiMn-LDH@PdNPs and an amino-modified ESAT-6 aptamer NH 2- Apt.

7. The preparation method of the nickel-manganese bimetallic layered nanoszyme probe according to claim 6, characterized in that, Comprising the following steps: dissolve NiMn-LDH@PdNPs to obtain a NiMn-LDH@PdNPs solution, mix the NiMn-LDH@PdNPs solution with an amino-modified ESAT-6 aptamer solution, ice-bath stir for 10-20 h, then wash, centrifuge, and dry the obtained precipitate C to obtain a nickel-manganese bimetallic layered nanoscale enzyme probe, denoted as NiMn-LDH@PdNPs-NH2-Apt.

8. Use of the nickel-manganese bimetallic layered nanoscale enzyme probe of claim 6 in the preparation of a product for detecting Mycobacterium tuberculosis antigen ESAT-6.

9. Use according to claim 8, characterized in that, The product is an electrochemical aptamer biosensor for detecting Mycobacterium tuberculosis antigen ESAT-6; The electrochemical aptamer biosensor comprises a gold nanoparticle modified electrode, streptavidin on the surface of the gold nanoparticle modified electrode, biotin-modified ESAT-6 ligand Bio-Apt, BSA for blocking non-specific sites on the electrode surface, and a nickel-manganese bimetallic layered nanoscale enzyme probe.

10. Use according to claim 9, characterized in that, The construction method of the electrochemical aptamer biosensor comprises the following steps: S1. Add streptavidin to the surface of the gold nanoparticle modified electrode, and incubate to obtain a streptavidin modified electrode; S2. Load biotin-modified ESAT-6 ligand Bio-Apt on the surface of the streptavidin modified electrode, and incubate to obtain a Bio-Apt modified electrode; S3. Load BSA on the surface of the Bio-Apt modified electrode to obtain an electrochemical aptamer biosensor substrate; S4. The surface of the electrochemical aptamer biosensor substrate is loaded with ESAT-6 antigen, incubated, reloaded with nickel-manganese bimetallic layered nanoscale enzyme probe, and incubated to obtain an electrochemical aptamer biosensor.

Images