Construction of a reusable nanopore-gated electrochemiluminescent sensor and its application in detecting T-2 toxin

Abstract

The present invention belongs to the technical field of electrochemiluminescent biosensing, and discloses a method for constructing a reusable nanopore-gated electrochemiluminescent sensor and its application in detecting T-2 toxin. The steps are: 1. Prepare a luminol / Au NPs composite material; 2. Prepare a luminol / Au NPs / CHI / GCE modified electrode; 3. Treat an AAO membrane; 4. Modify the detection area apt / Au-AAO; 5. Construct a nanopore-gated electrochemiluminescent aptamer sensor device for detecting T-2 toxin. 6. Reuse the device. The present invention utilizes an AAO membrane modified with Au NPs to regulate the diffusion of AA, separate the signal output area and the detection area, and test the electrochemiluminescence detection of the change in the luminescence intensity of the luminol / Au NPs luminophore. The present invention provides a detection mode that integrates the advantages of accuracy, rapidity, sensitivity, and reusability.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemiluminescent biosensing, and mainly relates to a construction method of a reusable nanopore-gated electrochemiluminescent sensor device and its application in detecting T-2 toxin. Background Art

[0002] Electrochemiluminescence (ECL) refers to the production of some special substances on the electrode surface through electrochemical methods. These substances form excited states through electron transfer between each other or with other components in the system, and then return to the ground state from the excited state to produce luminescence. It is a combination of electrochemical and chemiluminescence methods. It has the advantages of high sensitivity, wide linear range, simple instrumentation, strong controllability and good selectivity of chemiluminescence and electrochemistry. Common electrochemiluminescent bodies include luminol, ruthenium bipyridine, polycyclic aromatic hydrocarbons and other substances. Luminol (luminol) is a typical electrochemiluminescent body. Ascorbic acid (AA) can consume superoxide anion radicals (O2 ·- ) and active oxygen such as hydrogen peroxide to inhibit its ECL intensity.

[0003] Nanoporous materials refer to materials with a pore structure and a certain porosity with a significant surface effect and a pore size of 1 to 100 nm. They have controllable pores at the atomic, molecular and nanoscale, can distinguish molecules and clusters, and interact with them, and have good stability, good durability, good adsorption kinetics, high selectivity and high adsorption capacity. They are widely used in biosensors, drug delivery, gas separation, energy storage and fuel cell technology, nanocatalysis and photonics. As a nanoporous material, porous alumina (AAO) membrane has a stable structure, easy to control pore size, good biocompatibility, low preparation cost and reusability. It can be used in biosensors, DNA sensors, drug delivery and electrochemical sensors.

[0004] More than 100 mycotoxins have been isolated and identified so far. T-2 toxin, a toxic metabolite produced by the metabolism of Fusarium sporei, is widely distributed in cereal products and animal feed, and is stable and difficult to remove. Long-term contact between humans and animals can cause serious damage to body tissues, disorders, slow growth, and even death. The current detection methods for T-2 toxin include thin layer chromatography, high performance liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay, electrochemical analysis, etc. Although these methods are accurate, they have the disadvantages of long time, high cost, and complex operation, and are limited in practical application. Therefore, in view of this situation, it is very necessary to develop a simple, fast, and low-cost analysis method. Summary of the invention

[0005] The present invention aims to provide an electrochemiluminescent aptamer sensor that integrates the advantages of simple operation, high sensitivity, good selectivity, low cost, and reusability for the detection of T-2 toxin. The present invention connects an aptamer to an AAO membrane modified with Au NPs, uses Au-AAO nanopore gating to separate the signal output unit and the detection unit, and uses the nanomembrane mass transfer mechanism to adjust the actual pore size, and determines the change in the electrochemiluminescence intensity of luminol / Au NPs by affecting the diffusion of AA, thereby determining the concentration of T-2 toxin.

[0006] The method for constructing the electrochemiluminescence sensor device of the present invention comprises the following steps:

[0007] Step 1: Preparation of luminescent composite material luminol / Au NPs:

[0008] First, chloroauric acid solution was placed in a round-bottom flask and heated to boiling point, and then NaOH solution containing luminol was added thereto, and solution A was obtained under vigorous stirring; when the color of solution A changed from yellow to black and then to purple-red, the heat source was removed and cooled to room temperature, at which time the luminol / Au NPs material was prepared and stored at a certain temperature;

[0009] Step 2: Modify the signal output area:

[0010] The bare glassy carbon electrode was polished with alumina polishing powder until it became a mirror surface, and then ultrasonically washed in ethanol and deionized water, and dried for later use;

[0011] The product luminol / Au NPs obtained in step 1 was drop-coated on the surface of GCE, and then chitosan solution was added drop-wise to form a uniform film fixing material, which was naturally air-dried to obtain a luminol / Au NPs / CHI / GCE modified electrode;

[0012] Step 3: Processing AAO membrane:

[0013] The AAO membrane was ultrasonically cleaned in ethanol and deionized water in sequence, and then dried in a nitrogen stream to remove possible cutting debris on the surface of the AAO membrane;

[0014] Step 4: Modify the detection area:

[0015] Put the newly prepared Au NPs solution in a beaker, then add n-hexane, mix well, and continue to add anhydrous ethanol. After stratification, Au NPs can be seen to aggregate at the liquid-liquid interface. Then remove the n-hexane. Au NPs self-assemble and stabilize into a compact film at the gas-liquid interface. Carefully put the AAO membrane treated in step 3 into the surface of the solution in the beaker. The Au NPs membrane will automatically adsorb to the surface of the AAO membrane. After heating and drying, the Au-AAO nanofilm is obtained.

[0016] Then, a T-2 toxin aptamer solution was prepared using PBS as a solvent and added to the surface of the Au-AAO nanomembrane. After a period of reaction, the unbound aptamer was washed away with a PBS solution, and then an MCH solution was added to block the nonspecific active sites. Finally, the aptamer-modified detection area apt / Au-AAO was obtained.

[0017] Step 5: Construction of a nanopore-gated electrochemiluminescent aptamer sensor device for detecting T-2 toxin:

[0018] T-2 toxin solutions of different concentrations were dropped onto the detection area on apt / Au-AAO and incubated for a period of time to obtain T-2 / apt / Au-AAO;

[0019] Construction of the model system: The electrolytic cell of the model system consists of a left chamber and a right chamber, and the two chambers are separated by the T-2 / apt / Au-AAO nanomembrane obtained in step 5. The left chamber contains a PBS solution of H2O2 and the signal output area luminol / Au NPs / CHI / GCE modified in step 2, and the right chamber contains a PBS solution of AA and a Pt counter electrode and an Ag / AgCl reference electrode; and the Pt counter electrode and the Ag / AgCl reference electrode are placed on one side of the Au NPs membrane; using a three-electrode system, the electrochemiluminescence signal of luminol / Au NPs / CHI / GCE is detected to form a nanopore-gated electrochemiluminescence aptamer sensor device. In step 1, the volume ratio of the chloroauric acid solution to the NaOH solution containing luminol is 1:1, wherein the mass percentage concentration of the chloroauric acid solution is 0.01%, the concentration of luminol in the NaOH solution containing luminol is 0.01 g / mL, the concentration of NaOH is 0.1 mol / L, and the storage temperature is 4°C.

[0020] In step 2, the particle sizes of the aluminum oxide polishing powder are 1 μm, 0.3 μm and 0.05 μm;

[0021] The concentration of luminol / Au NPs solution was 5 mg / mL, and the drop-coated volume was 6 μL;

[0022] The mass percentage concentration of chitosan solution is 0.1%, and the drop amount is 5 μL;

[0023] In step 3, the ultrasonic time is 15 minutes, and the time for passing high-purity nitrogen is 10 minutes.

[0024] In step 4, the concentration of the Au NPs solution is 23.94 mmol / L; the particle size of the Au NPs solution is 20-25 nm;

[0025] The volume ratio of Au NPs solution, n-hexane, and anhydrous ethanol was 10 mL: 5 mL: 8 mL.

[0026] The heating and drying temperature is 70°C and the time is 2h.

[0027] The sequence of the T-2 toxin aptamer is: 5′-GTAT ATCA AGCA TCGC GTGT TTAC ACAT GCGA GAGGTGAA-SH-3′;

[0028] The concentration of T-2 toxin aptamer solution was 2 μmol / L, the drop volume was 20 μL, and the incubation time was 12 h;

[0029] The concentration of MCH solution was 1 mmol / L, and the amount added was 20 μL;

[0030] The pH of PBS solution is 7.4 and the concentration is 0.1 mol / L.

[0031] In step 5, the concentration of T-2 toxin is 0.005 ng / mL~10 6 ng / mL, the drop volume was 20 μL, the incubation temperature was 37 °C, and the incubation time was 45 min;

[0032] In the right chamber, in the PBS solution of AA, the concentration of AA is 0.02 mol / L, the pH of PBS is 7.4, and the concentration is 0.1 mol / L; in the left chamber, in the PBS solution of H2O2, the concentration of H2O2 is 10 mmol / L, the pH of PBS solution is 10, and the concentration is 0.1 mol / L; the volume of the solutions in the left and right chambers is equal.

[0033] The reusable nanopore-gated electrochemiluminescent aptamer sensor device prepared by the present invention is used to detect T-2 toxin, and the specific steps are as follows:

[0034] (1) dropping different concentrations of T-2 toxin solutions onto the detection area on apt / Au-AAO and incubating for a period of time to obtain T-2 / apt / Au-AAO;

[0035] (2) Constructing a model system: The electrolytic cell of the model system consists of a left chamber and a right chamber, and the two chambers are separated by the T-2 / apt / Au-AAO nanomembrane obtained in step (1). The left chamber contains a PBS solution of H2O2 and the modified signal output area luminol / Au NPs / CHI / GCE in step 2, and the right chamber contains a PBS solution of AA and a Pt counter electrode and an Ag / AgCl reference electrode; and the Pt counter electrode and the Ag / AgCl reference electrode are placed on one side of the Au NPs membrane; a three-electrode system is used to connect to an MPI-A electrochemiluminescence detector, and a potential scan is performed, with the luminol / Au NPs luminescence intensity as the output signal; the ECL value when the luminol / Au NPs are luminescent and the T-2 toxin concentration are used as a standard curve;

[0036] (3) Using the above method to collect signals from a T-2 toxin solution of unknown concentration, substitute the signals into the standard curve to obtain the concentration of the T-2 toxin solution.

[0037] (4) Take out the T-2 / apt / Au-AAO nanomembrane from the sensor device after the T-2 toxin detection, immerse it in ultrapure water, and place it in an oven to open the aptamer fixed by the thiol group; then rinse it with PBS, add the unknown concentration of T-2 toxin to apt / Au-AAO, repeat step (3), and obtain the concentration of T-2 toxin solution.

[0038] In step (1), the concentration of T-2 toxin is 0.005 ng / mL to 10 6 ng / mL, the drop volume was 20μL; the incubation temperature was 37℃, and the incubation time was 45min;

[0039] In step (2), in the right chamber, the concentration of AA in the PBS solution is 0.02 mol / L, the pH of PBS is 7.4, and the concentration is 0.1 mol / L; in the left chamber, the concentration of H2O2 in the PBS solution is 10 mmol / L, the pH of PBS solution is 10, and the concentration is 0.1 mol / L; the volumes of the solutions in the left and right chambers are equal.

[0040] The scanning potential was 0.2-0.7 V, and the reaction time was 600 s.

[0041] In step (4), the oven temperature is 90° C.; the pH of the PBS solution is 7.4, and the concentration is 0.1 mol / L.

[0042] The beneficial effects of the present invention are:

[0043] The present invention successfully established a reusable nanopore-gated electrochemiluminescent aptamer sensor by modifying the luminol / Au NPs region on the glassy carbon electrode as the signal output region and modifying apt on the Au-AAO as the detection region for quantitative detection of T-2 toxin. Its features and advantages are described as follows:

[0044] (1) The present invention prepares luminol / Au NPs as a luminophore, modifies it on a glassy carbon electrode as a signal output region, and uses apt / Au-AAO as a detection region to construct a nanopore-gated electrochemiluminescence sensor, and the luminescence signal is stable.

[0045] (2) The present invention prepares luminol / Au NPs to enhance the luminescence of luminol, and based on the present invention, the detection of T-2 toxin can be realized. Differentiating the detection area and the signal output area can reduce the contamination of the aptamer and toxin to the luminescent material and affect its luminescence.

[0046] (3) The electrochemiluminescent aptasensor proposed in the present invention achieves sensitive detection of T-2 toxin at a concentration of 0.01 ng / mL to 10 5 In the range of ng / mL concentration logarithm, the T-2 toxin concentration and the ECL value of the luminol / Au NPs luminescence intensity showed a good linear relationship, and the detection limit could reach 0.0033 ng / mL.

[0047] (4) The nanopore-gated electrochemiluminescent aptamer sensor constructed by the present invention can achieve highly sensitive quantitative detection, and has the advantages of short detection process, small amount of reagents, low detection cost, and reusability. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Diagram of the detection mechanism of the constructed reusable nanopore-gated electrochemiluminescent aptamer sensor;

[0049] Figure 2 A is the X-ray diffraction pattern of Au NPs, and B is the UV-visible absorption spectrum;

[0050] Figure 3 Transmission electron microscopy image of Au NPs;

[0051] Figure 4 A is a scanning electron microscope image of an AAO film, and B is a scanning electron microscope image of an Au-AAO nanofilm;

[0052] Figure 5A is the linear relationship diagram between the lg value of different T-2 toxin concentrations and the luminescence intensity of the luminol / Au NPs composite material, B is the result diagram of the sensor selectivity test, and C is the result diagram of the cyclic test reusability. DETAILED DESCRIPTION

[0053] The present invention is described in detail below with reference to the accompanying drawings and examples, but the present invention is not limited to these embodiments.

[0054] Figure 1 Diagram of the detection mechanism of the constructed reusable nanopore-gated electrochemiluminescent aptamer sensor.

[0055] (1) Preparation of composite luminol / Au NPs:

[0056] Take 20mL of 0.01% chloroauric acid solution and place it in a round-bottom flask, heat it to boiling point; then add 20mL of luminol solution with a concentration of 10mg / mL previously dissolved in NaOH, and stir vigorously for 30min. During this period, the color is observed to gradually change from yellow to black, and finally to wine red or purple. This indicates that the luminol / Au NPs solution has been formed. At this time, remove the heat source, cool the colloid to room temperature, and then store it at 4°C.

[0057] (2) Modify the signal output area:

[0058] Before modification, the bare glassy carbon electrode was polished with alumina polishing powder with particle sizes of 1 μm, 0.3 μm and 0.05 μm in sequence until it became a mirror surface, and then ultrasonically washed in ethanol and deionized water for one minute and dried for later use.

[0059] 6 μL of luminol / Au NPs composite material was drop-coated on the treated glassy carbon electrode surface and dried at room temperature to form a luminol / Au NPs modified electrode. Then 5 μL of 0.1% chitosan solution was modified on the electrode to form a uniform film fixing material, which was naturally air-dried to obtain a modified nanocomposite electrode, which was recorded as luminol / Au NPs / CHI / GCE.

[0060] (3) AAO membrane separator pretreatment:

[0061] The AAO membrane was ultrasonically cleaned in ethanol and deionized water in sequence and then dried in a nitrogen stream to remove possible cutting debris on the surface of the AAO membrane.

[0062] (4) Modify the detection area:

[0063] Put 10mL of Au NPs solution in a beaker, add 5mL of n-hexane and mix well. Then add 8mL of anhydrous ethanol. After stratification, Au NPs can be seen to aggregate at the liquid-liquid interface. Then remove the n-hexane, and the Au NPs self-assemble and stabilize into a compact film at the gas-liquid interface. Carefully place the AAO membrane on the surface of the solution in the beaker, and the Au NPs membrane will automatically adsorb to the surface of the AAO membrane. After heating and drying, an Au-AAO nanomembrane is formed, that is, Au-AAO nanopore gating.

[0064] Then, a 2 μmol / L T-2 toxin aptamer solution was prepared using PBS at pH = 7.4 and 0.1 mol / L as solvent. The T-2 aptamer sequence was: 5'-GTAT ATCA AGCA TCGC GTGT TTAC ACAT GCGA GAGG TGAA-SH-3'. A volume of 20 μL of T-2 toxin aptamer solution was added dropwise to Au-AAO, placed in a 4°C refrigerator for reaction for 12 hours, and rinsed several times with PBS buffer solution at pH = 7.4 to remove the unadsorbed aptamer. Then, 20 μL of 1 mmol / L MCH solution was added dropwise to block the nonspecific active sites, and the aptamer-modified detection region apt / Au-AAO was obtained.

[0065] (5) Construction of a nanopore-gated electrochemiluminescent aptamer sensor device for detecting T-2 toxin:

[0066] 20 μL of T-2 toxin solution with concentrations of 0.005 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 10000 ng / mL, 100000 ng / mL, and 1000000 ng / mL were respectively added to the detection area of ​​apt / Au-AAO and incubated at 37°C for 45 min to obtain T-2 / apt / Au-AAO; the model system was constructed: the electrolytic cell of the model system consisted of a left chamber and a right chamber, the two chambers were separated by a T-2 / apt / Au-AAO nanomembrane, the left chamber contained a PBS solution of H2O2 and the signal output area modified in step 2, luminol / Au NPs / CHI / GCE, and the right chamber contained a PBS solution of AA and a Pt counter electrode and an Ag / AgCl reference electrode; and the Pt counter electrode and the Ag / AgCl reference electrode were placed on the Au On one side of the NPs membrane; a three-electrode system was used to connect the MPI-A electrochemiluminescence detector for potential scanning, with a potential of 0.2-0.7V and a reaction time of 600s. The luminol / Au NPs luminescence intensity was used as the output signal; by analyzing the changes in the ECL signal value when the luminol / Au NPs were luminescent, a reusable nanopore-gated electrochemiluminescent aptamer sensor device could be constructed. In the right chamber, the concentration of ascorbic acid (AA) was 0.02mol / L, the volume of the PBS solution was 10mL, the pH was 7.4, and the concentration was 0.1mol / L;

[0067] In the left ventricle, the H2O2 concentration in the PBS solution was 10mmol / L, the volume of the PBS solution was 10mL, the pH was 10, and the concentration was 0.1mol / L;

[0068] The characterization results are shown in the figure. Figure 2 , Figure 3 It can be seen that Au NPs with a particle size of about 20-25 nm have been successfully prepared; Figure 4 It can be seen that Au NPs have been successfully loaded on the AAO membrane;

[0069] from Figure 5 It can be seen from A that in the range of 0.01ng / mL to 10000ng / mL concentration logarithm, the ECL value of luminol / Au NPs during luminescence shows a good linear relationship with the lg value of T-2 toxin concentration, and the detection limit can reach 0.0033ng / mL.

[0070] (6) Reusing the nanopore-gated electrochemiluminescent aptamer sensor device for detecting T-2 toxin to detect T-2 toxin:

[0071] The T-2 / apt / Au-AAO nanomembrane was taken out from the sensor device after the T-2 toxin detection was completed, immersed in ultrapure water, and placed in a 90°C oven to open the aptamer fixed by the thiol group; it was then rinsed with PBS with a pH of 7.4 and a concentration of 0.1 mol / L, and then T-2 toxin with a concentration of 10 ng / mL was added to apt / Au-AAO, and repeated (5) to test the ECL signal of the luminol / AuNPs / CHI / GCE modified electrode.

[0072] Test results such as Figure 5 ,from Figure 5 From B, we can see that the sensor has good selectivity; Figure 5 It can be seen from the C that the sensor can be reused after multiple cycle tests.

Claims

1. A method for constructing a reusable nanopore-gated electrochemiluminescent sensor, characterized in that: The following steps are involved: Step 1: Preparation of luminescent composite material luminol / Au NPs: First, chloroauric acid solution was placed in a round-bottom flask and heated to boiling point, and then NaOH solution containing luminol was added thereto, and solution A was obtained under vigorous stirring; when the color of solution A changed from yellow to black and then to purple-red, the heat source was removed and cooled to room temperature, at which time the luminol / Au NPs material was prepared and stored at a certain temperature; Step 2: Modify the signal output area: The bare glassy carbon electrode was polished with alumina polishing powder until it became a mirror surface, and then ultrasonically washed in ethanol and deionized water, and dried for later use; The product luminol / Au NPs obtained in step 1 was drop-coated on the surface of GCE, and then chitosan solution was added drop-wise to form a uniform film fixing material, which was naturally air-dried to obtain a luminol / Au NPs / CHI / GCE modified electrode; Step 3: Processing AAO membrane: The AAO membrane was ultrasonically cleaned in ethanol and deionized water in sequence, and then dried in a nitrogen stream to remove possible cutting debris on the surface of the AAO membrane; Step 4: Modify the detection area: Put the newly prepared Au NPs solution in a beaker, then add n-hexane, mix well, and continue to add anhydrous ethanol. After stratification, Au NPs can be seen to aggregate at the liquid-liquid interface. Then remove the n-hexane. Au NPs self-assemble and stabilize into a compact film at the gas-liquid interface. Carefully put the AAO membrane treated in step 3 into the surface of the solution in the beaker. The Au NPs membrane will automatically adsorb to the surface of the AAO membrane. After heating and drying, the Au-AAO nanofilm is obtained. Then, a T-2 toxin aptamer solution was prepared using PBS as a solvent and added to the surface of the Au-AAO nanomembrane. After a period of reaction, the unbound aptamer was washed away with a PBS solution, and then an MCH solution was added to block the nonspecific active sites. Finally, the aptamer-modified detection area apt / Au-AAO was obtained. Step 5: Construction of a nanopore-gated electrochemiluminescent aptamer sensor device for detecting T-2 toxin: T-2 toxin solutions of different concentrations were dropped onto the detection area on apt / Au-AAO and incubated for a period of time to obtain T-2 / apt / Au-AAO; Construction of the model system: The electrolytic cell of the model system consists of a left chamber and a right chamber. The two chambers are separated by the aptamer-modified T-2 / apt / Au-AAO nanomembrane obtained in step 5. The left chamber contains a PBS solution of H2O2 and the signal output area luminol / Au NPs / CHI / GCE modified in step 2, and the right chamber contains a PBS solution of AA and a Pt counter electrode and an Ag / AgCl reference electrode; and the Pt counter electrode and the Ag / AgCl reference electrode are placed on one side of the Au NPs membrane; using a three-electrode system, the electrochemiluminescence signal of luminol / AuNPs / CHI / GCE is detected to form a nanopore-gated electrochemiluminescence aptamer sensor device.

2. The construction method according to claim 1, characterized in that: In step 1, the volume ratio of the chloroauric acid solution to the NaOH solution containing luminol is 1:1, wherein the mass percentage concentration of the chloroauric acid solution is 0.01%, the concentration of luminol in the NaOH solution containing luminol is 0.01 g / mL, the concentration of NaOH is 0.1 mol / L, and the storage temperature is 4°C.

3. The construction method according to claim 1, characterized in that: In step 2, The particle sizes of alumina polishing powder are 1μm, 0.3μm and 0.05μm. The concentration of luminol / Au NPs solution was 5 mg / mL, and the drop-coated volume was 6 μL; The mass percentage concentration of the chitosan solution was 0.1%, and the drop amount was 5 μL.

4. The construction method according to claim 1, characterized in that: In step 3, The ultrasonic time was 15 min, and the time for passing high-purity nitrogen was 10 min.

5. The construction method according to claim 1, characterized in that: In step 4, The concentration of Au NPs solution is 23.94mmol / L; the particle size of Au NPs solution is 20-25nm; The volume ratio of Au NPs solution, n -hexane, and anhydrous ethanol was 10 mL:5 mL:8 mL; The heating and drying temperature is 70°C and the time is 2h.

6. The construction method according to claim 1, characterized in that: In step 4, The sequence of the T-2 toxin aptamer is: 5′-GTAT ATCA AGCA TCGC GTGT TTAC ACAT GCGA GAGG TGAA-SH-3′; The concentration of T-2 toxin aptamer solution was 2 μmol / L, the drop volume was 20 μL, and the incubation time was 12 h; The concentration of MCH solution was 1 mmol / L, and the amount added was 20 μL; The pH of PBS solution is 7.4 and the concentration is 0.1 mol / L.

7. The construction method according to claim 1, characterized in that: In step 5, the concentration of T-2 toxin is 0.005 ng / mL~10 6 ng / mL, the drop volume was 20 μL, the incubation temperature was 37 °C, and the incubation time was 45 min; In the right chamber, the concentration of AA in PBS solution was 0.02 mol / L, the pH of PBS was 7.4, and the concentration was 0.1 mol / L; In the left ventricle, the H2O2 concentration in the PBS solution was 10mmol / L, the pH of the PBS solution was 10, and the concentration was 0.1mol / L; The volume of solution in the left and right chambers is equal.

8. Use of the reusable nanopore-gated electrochemiluminescent sensor constructed by the construction method according to any one of claims 1 to 7 for detecting T-2 toxin.

9. The use according to claim 8, characterized in that The specific steps are: (1) dropping different concentrations of T-2 toxin solutions onto the detection area on apt / Au-AAO and incubating for a period of time to obtain T-2 / apt / Au-AAO; (2) Constructing a model system: The electrolytic cell of the model system consists of a left chamber and a right chamber, and the two chambers are separated by the T-2 / apt / Au-AAO nanomembrane obtained in step (1). The left chamber contains a PBS solution of H2O2 and the modified signal output area luminol / Au NPs / CHI / GCE in step 2, and the right chamber contains a PBS solution of AA and a Pt counter electrode and an Ag / AgCl reference electrode; and the Pt counter electrode and the Ag / AgCl reference electrode are placed on one side of the Au NPs membrane; a three-electrode system is used to connect to an MPI-A electrochemiluminescence detector, and a potential scan is performed, with the luminol / Au NPs luminescence intensity as the output signal; the ECL value when the luminol / Au NPs are luminescent and the T-2 toxin concentration are used as a standard curve; (3) using the above method to collect signals of a T-2 toxin solution of unknown concentration, and substituting the signals into the standard curve to obtain the concentration of the T-2 toxin solution; (4) Take out the T-2 / apt / Au-AAO nanomembrane from the sensor device after the T-2 toxin detection, immerse it in ultrapure water, and place it in an oven to open the aptamer fixed by the thiol group; then rinse it with PBS, add the unknown concentration of T-2 toxin to apt / Au-AAO, repeat step (3), and obtain the concentration of T-2 toxin solution.

10. The use according to claim 9, characterized in that In step (1), the concentration of T-2 toxin is 0.005 ng / mL to 10 6 ng / mL, the drop volume was 20μL; the incubation temperature was 37℃, and the incubation time was 45min; In step (2), in the right chamber, in the PBS solution of AA, the concentration of AA is 0.02 mol / L, the pH of PBS is 7.4, and the concentration is 0.1 mol / L; in the left chamber, in the PBS solution of H2O2, the concentration of H2O2 is 10 mmol / L, the pH of PBS solution is 10, and the concentration is 0.1 mol / L; the volumes of the solutions in the left and right chambers are equal; The scanning potential was 0.2-0.7 V, and the reaction time was 600 s; In step (4), the oven temperature is 90° C.; the pH of the PBS solution is 7.4, and the concentration is 0.1 mol / L.

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