Electrochemical immunosensor and detection method for zearalenone detection

Abstract

The present invention provides an electrochemical immunosensor for detecting zearalenone (ZEA). Using TB as the electroactive substance, PDDA-NG / HNT is used as the carrier to improve the stability of TB, and AuNPs are used to connect antibodies, showing a strong signal amplification effect. In the present invention, Ti-MOF / KB is dropped with AuNPs as the sensing interface. On the one hand, it improves the conductive activity of the electrode and increases the specific surface area of the electrode. On the other hand, it can capture a large amount of primary antibody Ab1, and specifically binds with the signal probe through Ab1-Ag-Ab2 to construct a sandwich-type sensor, realizing the highly sensitive detection of zearalenone. The detection method of the present invention has the advantages of a wide linear range, strong specificity, short analysis time, good stability and reproducibility, and is suitable for application in many fields.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical detection, and in particular to an electrochemical immunosensor and a detection method for detecting zearalenone. Background Art

[0002] Among food contaminants, mycotoxins are secondary metabolites of fungi that are toxic to humans and animals. Zearalenone (ZEA) is a mycotoxin produced by different filamentous fungi such as Fusarium graminearum, Fusarium solani and Fusarium kluyveri. ZEA is found in cereals, mainly corn, and processed foods. It is one of the most widely distributed mycotoxins in the world, with a lethal dose of 4000 to 10000 mg / kg of body weight. In addition, long-term exposure to ZEA can cause reproductive disorders, estrogenicity, abortion and infertility, as well as hepatotoxic, immunotoxic and hematotoxic effects. In Argentina, the maximum content of zearalenone allowed in corn is 200 μg / kg. Therefore, the detection of zearalenone in food is of great significance.

[0003] At present, high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis, surface plasmon resonance, solid phase extraction and other methods have been used to detect zearalenone. These analytical methods are sensitive and selective, but due to the bulky instruments, they are not suitable for on-site monitoring and analysis. In addition, these methods are expensive, require well-trained skilled personnel to operate, and require lengthy sample preparation and cleanup procedures. Therefore, it is necessary to find a fast, simple, convenient and accurate method for the detection of zearalenone. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides an electrochemical immunosensor for detecting zearalenone (ZEA).

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

[0006] An electrochemical immunosensor for detecting zearalenone (ZEA), characterized in that:

[0007] The method for constructing an electrochemical immunosensor for detecting zearalenone (ZEA) is as follows: a Ti-MOF / KB solution is dripped onto a clean glassy carbon electrode surface, and then an AuNPs solution is dripped onto the electrode surface after drying at room temperature, and then the solution is dried at room temperature, and Ab1 is dripped onto the electrode, and the solution is incubated overnight in a refrigerator at 4-5° C. for 12-13 hours; after being rinsed with DEPC water, a 1% bovine serum albumin (BSA) solution is dripped, and the solution is incubated at 4-5° C. for 30-35 minutes; finally, the obtained electrode is rinsed with DEPC water, and a PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution is dripped, and the solution is incubated at room temperature for 4-5 hours, so as to obtain an electrochemical immunosensor for detecting zearalenone (ZEA);

[0008] Preparation of the PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution: adding the zearalenone polyclonal antibody Ab2 to the prepared PDDA-NG / HNT / TB-AuNPs dispersion, stirring in an ice bath for 12-13 hours, centrifuging, washing with water, and redispersing the precipitate in ultrapure water to obtain the PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution.

[0009] The preparation method of the PDDA-NG / HNT / TB-AuNPs dispersion is as follows: the prepared AuNPs are added to the prepared PDDA-NG / HNT / TB dispersion, stirred for 4-5 hours, centrifuged, washed, and then the precipitate is dispersed in ultrapure water to obtain the PDDA-NG / HNT / TB-AuNPs dispersion.

[0010] The PDDA-NG / HNT / TB dispersion is prepared by adding toluidine blue (TB) to the prepared PDDA-NG / HNT dispersion, stirring for 12-13 hours, centrifuging, washing, and dispersing the precipitate in ultrapure water to obtain the PDDA-NG / HNT / TB dispersion.

[0011] The preparation method of the PDDA-NG / HNT dispersion is as follows: dissolving nitrogen-doped graphene (NG) in N,N-dimethylformamide (DMF), adding polydiallyl dimethyl ammonium chloride (PDDA) and stirring for 2-3 hours, taking the solution and adding halloysite nanotubes (HNT) and stirring for 3 hours to obtain the PDDA-NG / HNT dispersion.

[0012] The preparation method of the Ti-MOF / KB dispersion is as follows: 2-aminoterephthalic acid (NH2-BDC) is added to a N,N-dimethylformamide (DMF)-methanol mixed solvent, and then tetrabutyl titanate and Ketjen black (KB) are added, and after ultrasonic treatment at room temperature for 10-15 minutes, the solution is transferred to a reactor and reacted at 150-160° C. for 20-24 hours, cooled, centrifuged at 10,000 rpm for 5-6 minutes, washed with DMF and methanol, and dried in an oven at 60-70° C. to obtain a dark green Ti-MOF / KB nanomaterial; and then dissolved with ultrapure water to obtain a Ti-MOF / KB dispersion.

[0013] The preparation method of the AuNPs is as follows: adding 1% HAuCl4 solution to ultrapure water and boiling, then adding 1% trisodium citrate solution and continuing to boil for 15 minutes, cooling and restoring the volume to the original volume with ultrapure water to obtain a transparent wine red solution, which is the gold nanoparticles (AuNPs).

[0014] An electrochemical immunosensor for detecting zearalenone, characterized in that the method for constructing an electrochemical immunosensor for detecting zearalenone comprises the following steps:

[0015] (1) Preparation of signal probe:

[0016] 1) PDDA-NG / HNT: 10 mg of nitrogen-doped graphene (NG) was dissolved in 10 mL of N,N-dimethylformamide (DMF), 4 mL of polydiallyl dimethyl ammonium chloride (PDDA) was added and stirred for 2 h, 2 mL of the above solution was added to halloysite nanotubes (HNT) (1 mg; 2 mg; 3 mg; 4 mg; 5 mg) and stirred for 3 h to obtain a PDDA-NG / HNT dispersion;

[0017] 2) PDDA-NG / HNT / TB: 1 mL of the prepared PDDA-NG / HNT dispersion was added with 1 mL of 1 mg / mL toluidine blue (TB) and stirred for 12 h. After centrifugation and washing with ultrapure water, the black precipitate was dispersed in 1 mL of ultrapure water to obtain a PDDA-NG / HNT / TB dispersion;

[0018] 3) AuNPs: Add 1 mL of 1% HAuCl4 solution to 100 mL of ultrapure water and boil, then quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 minutes. After cooling, restore the volume to the original volume with ultrapure water to obtain a transparent wine red solution, which is gold nanoparticles (AuNPs);

[0019] 4) PDDA-NG / HNT / TB-AuNPs: 500 μL of the prepared AuNPs was added to 1 mL of the prepared PDDA-NG / HNT dispersion material, stirred for 4 h, centrifuged, washed with ultrapure water, and the black precipitate was dispersed in 1 mL of ultrapure water to obtain a PDDA-NG / HNT / TB-AuNPs dispersion;

[0020] 5) PDDA-NG / HNT / TB-AuNPs / Ab2: Add 200 μL of 10 μg / mL Ab2 to 1 mL of the prepared PDDA-NG / HNT / TB-AuNPs dispersion, stir in an ice bath for 12 h, centrifuge, wash with water, and redisperse the precipitate in 1 mL of ultrapure water to obtain the PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution.

[0021] (2) Preparation of substrate materials

[0022] Ti-MOF / KB dispersion: 3.1 mmol 2-aminoterephthalic acid (NH2-BDC) was added into 15 mL N,N-dimethylformamide (DMF)-methanol mixed solvent (V DMF :V 甲醇＝ 9:1), then add 2.0mmol tetrabutyl titanate and 10mg Ketjen black (KB), ultrasonicate at room temperature for 10min, transfer the above solution to a reactor and react at 150℃ for 24h, cool, centrifuge at 10000rpm for 5min, wash with DMF and methanol, and dry in an oven at 60℃ to obtain dark green Ti-MOF / KB nanomaterials; take 1mg of the above material and dissolve it in 1mL ultrapure water to obtain Ti-MOF / KB dispersion.

[0023] (3) Construction of an electrochemical immunosensor for detecting zearalenone:

[0024] 1) treating the capture antibody Ab1 and the zearalenone antigen with 0.1M PBS (pH=7) buffer at room temperature and storing them for later use;

[0025] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2=3:1, v / v) for 30 min and then rinse with ultrapure water for later use;

[0026] 3) The electrode obtained in step 2) was polished to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders, and then the electrode was ultrasonically treated in the order of ultrapure water, anhydrous ethanol, and ultrapure water, and dried for later use;

[0027] 4) electrochemically activating the electrode obtained in step 3) in 0.5M H2SO4, then rinsing with ultrapure water and drying;

[0028] 5) 10 μL of Ti-MOF / KB dispersion was added dropwise onto the surface of the cleaned glassy carbon electrode in step 4), and after drying at room temperature, 10 μL of AuNPs was added dropwise onto the surface of the electrode and dried at room temperature;

[0029] 6) 10 μL of Ab1 prepared in step 1) was dripped onto the electrode prepared in step 5) and incubated overnight in a refrigerator at 4°C for 12 h;

[0030] 7) After the electrode obtained in step 6) is rinsed with DEPC water, 5 μL of 1% bovine serum albumin (BSA) solution is added and incubated at 4°C for 30 min;

[0031] 8) After the electrode obtained in step 7) is rinsed with DEPC water, different concentrations of zearalenone are added dropwise to the electrode surface and incubated at 4°C for 1 hour;

[0032] 9) After the electrode obtained in step 8) is rinsed with DEPC water, 10 μL of PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution is added dropwise and incubated at room temperature for 4 h to obtain an electrochemical immunosensor for the detection of zearalenone.

[0033] The present invention also provides a method for detecting zearalenone by using the electrochemical immunosensor.

[0034] A method for detecting zearalenone using the electrochemical immunosensor, characterized in that it comprises the following steps:

[0035] 1) adding different concentrations of target zearalenone to the sensor electrode;

[0036] 2) placing the electrode in a 0.1 M PBS (pH = 7.0) solution for characterization and measuring its electrochemical signal;

[0037] 3) drawing a working curve according to the linear relationship between the current value obtained in step 2) and the logarithmic value of the zearalenone concentration;

[0038] 4) The sample to be tested is detected by the sensor, and the obtained current value is calculated through the working curve prepared in step 3) to obtain the concentration of zearalenone in the sample to be tested.

[0039] Compared with the prior art, the preparation method and application of the electrochemical immunosensor for detecting zearalenone of the present invention have the following outstanding features:

[0040] The present invention prepares a composite material of nitrogen-doped graphene and halloysite nanotubes functionalized with polydimethyl ammonium chloride (PDDA), loads electroactive substance toluidine blue (TB) through π-π stacking and adsorption, and is modified with gold nanoparticles (AuNPs). A large amount of secondary antibody Ab2 is loaded through the bonding action of metal particles and amino groups, and finally a PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution is prepared. TB tends to aggregate in a solvent to form a polymer, and the polymer reduces its electron transfer ability and stability. The inventor uses the composite material of nitrogen-doped graphene and halloysite nanotubes functionalized with PDDA as a carrier, so that more electroactive substances are adsorbed on the surface, the stability of TB is increased, the electrical signal of the sensor is amplified, and its sensitivity is improved. In addition, the inventor uses Ti-MOF / KB dripping AuNPs as a sensing platform to capture a large amount of primary antibody Ab1 to further achieve signal amplification. The present invention utilizes the large specific surface area and good conductivity of nitrogen-doped graphene (NG) to provide abundant binding sites for TB. At the same time, halloysite nanotubes have low cost, non-toxicity, hollow tubular structure, large specific surface area, high thermal stability and mechanical properties, and strong adsorption capacity for TB. This work synthesized for the first time a composite material of nitrogen-doped graphene and halloysite nanotubes functionalized with PDDA as a carrier, so that more electroactive substances are adsorbed on the surface, the stability of TB is increased, the electrical signal of the sensor is amplified, and its sensitivity is improved. In addition, the present invention firstly incorporates Ketjen black (KB) into Ti-MOF, and uses it as a substrate to improve the conductive activity of the electrode and increase the specific surface area of ​​the electrode. By dripping gold nanoparticles on the substrate material to capture a large amount of primary antibody Ab1, signal amplification is further achieved. By the above means, the prepared electrochemical immunosensor is successfully used for ultrasensitive detection of zearalenone. Compared with the traditional detection method, the advantages of the present invention are high sensitivity, strong specificity, rapid detection, convenient operation, low price of equipment and materials, and no pollution, thereby providing a new detection method for the detection of zearalenone.

[0041] Beneficial effects of the present invention:

[0042] 1) By using a composite material of PDDA-functionalized nitrogen-doped graphene and halloysite nanotubes as a carrier, the characteristics of TB's easy aggregation and poor stability can be improved, thereby improving the sensor performance and achieving signal amplification, while also improving the detection sensitivity.

[0043] 2) Ti-MOF / KB is added with gold nanoparticles as the sensing interface, which increases the conductivity of the electrode on the one hand, and can immobilize more capture probes on the other hand, and construct a sandwich sensor with the signal probe through Ab1-Ag-Ab2 specific binding, and realizes high-sensitivity detection of zearalenone through the signal generated by TB. In addition, the biosensor prepared by the present invention also has the advantages of wide linear range, strong specificity, short analysis time, good stability and reproducibility, etc.

[0044] 3) The biosensor of the present invention has a high degree of specificity in identifying the target, which can improve the selectivity of the sensor, thereby providing a new research direction and analysis method for the detection of trace amounts of zearalenone.

[0045] 4) The materials involved in the present invention can all be synthesized under laboratory conditions, with simple operation, low raw material price, low toxicity, environmental friendliness, and extremely small amount used each time, thus reducing experimental costs.

[0046] 5) The electrochemical immunosensor prepared by this method can provide a new method for the detection of zearalenone; the electrochemical immunosensor prepared by this invention can also be applied to other biological sample determinations, food, drug and environmental monitoring, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the calibration curve of the logarithmic value of zearalenone at different concentrations and the DPV response current value of the sensor.

[0048] Figure 2 The long-term stability test results of the sensor obtained after incubation with 100 ng / mL ZEA for 14 days.

[0049] Figure 3 This is the reproducibility result of the sensors obtained by incubating 1pg / mL ZEA with five different glassy carbon electrodes at the same time and scanning under the same conditions.

[0050] Figure 4 Cyclic voltammetry characterization diagrams of different modified electrodes obtained in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution with a voltage range from -0.6 to 0.2 V and a scan rate of 100 mV / s. DETAILED DESCRIPTION

[0051] Additional aspects and advantages of the present invention will be described in part in the following description, and will become apparent from the following description or learned through practice of the present invention. The present invention will be further described below in conjunction with the embodiments, but the present invention is not limited thereto.

[0052] The main chemical reagents used in the embodiments of the present invention are as follows:

[0053] Ketjen black (KB) was purchased from Lion Corporation, Japan; zearalenone (ZEA) antigen and polyclonal antibody were purchased from Shanghai Bio-Technology Co., Ltd.; toluidine blue (TB) was purchased from Beijing Bailingwei Technology Co., Ltd.; tetrabutyl titanate and N,N-dimethylformamide (DMF) were purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China). Nitrogen-doped graphene (NG) was purchased from Xianfeng Nanomaterials Technology Co., Ltd. Bovine serum albumin (BSA) was purchased from J&K Scientific Ltd (Beijing, China); 2-aminoterephthalic acid (NH2-BDC), polydimethylammonium chloride (PDDA) and chloroauric acid (HAuCl4) were purchased from Sigma (USA). Methanol was purchased from Chuandong Chemical Group Co., Ltd.

[0054] Equipment and technical parameters used:

[0055] Instrumentation: Electrochemical detection was performed using a three-electrode system: a modified glassy carbon electrode (4 mm in diameter) was used as the working electrode, a platinum wire was used as the counter electrode, and a saturated calomel electrode (SCE) was used as the reference electrode. Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were performed using a Metrohm Autolab BV electrochemical workstation (Modular Instruments, Switzerland). pH was monitored by a pH meter (MP 230, Mettler-Toledo, Switzerland). Cyclic voltammetry (CV) curves were scanned at 100 mV / s in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution by an electrochemical three-electrode system. Differential pulse voltammetry (DPV) curves were scanned from -0.5 to 0.1 in 0.1 M PBS (pH 7.0) by an electrochemical three-electrode system at a scan rate of 50 mV / s.

[0056] Example 1

[0057] Follow these steps:

[0058] (1) Preparation of signal probes;

[0059] 1) PDDA-NG / HNT: Dissolve 10 mg of nitrogen-doped graphene (NG) in 10 mL of N,N-dimethylformamide (DMF), add 4 mL of 5% polydimethylammonium chloride (PDDA) and stir on a magnetic stirrer for 2 h to obtain PDDA-functionalized nitrogen-doped graphene solution. Take 2 mL of the above solution and add halloysite nanotubes (HNT) (1 mg; 2 mg; 3 mg; 4 mg; 5 mg) respectively and stir for 3 h to obtain PDDA-NG / HNT dispersion.

[0060] 2) PDDA-NG / HNT / TB: Take 1 mL of the prepared PDDA-NG / HNT dispersion, add 1 mL of 1 mg / mL toluidine blue (TB), stir overnight for 12 h, centrifuge at 12,000 rpm for 5 min, wash three times with ultrapure water, and then disperse the black precipitate in 1 mL of ultrapure water to obtain a PDDA-NG / HNT / TB dispersion.

[0061] 3) AuNPs: Add 1 mL of 1% HAuCl4 solution to 100 mL of ultrapure water and boil, then quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 minutes. After cooling, restore the volume to the original volume with ultrapure water to obtain a transparent wine-red solution, which is gold nanoparticles (AuNPs).

[0062] 4) PDDA-NG / HNT / TB-AuNPs: Take 500 μL of AuNPs prepared in step 3) and add it to 1 mL of PDDA-NG / HNT / TB dispersion material prepared in step 2), stir it at room temperature for 4 h on a magnetic stirrer, centrifuge it at 10,000 rpm for 5 min, wash it three times with ultrapure water, and disperse the black precipitate in 1 mL of ultrapure water to obtain a PDDA-NG / HNT / TB-AuNPs dispersion.

[0063] 5) PDDA-NG / HNT / TB-AuNPs / Ab2: Add 200 μL of 10 μg / mL zearalenone polyclonal antibody Ab2 to 1 mL of the PDDA-NG / HNT / TB-AuNPs dispersion prepared in step 4), and stir in an ice bath for 12 h to fully anchor it on the surface of the composite material to form a signal probe. After centrifugation and washing, the complex was redissolved in 1 mL of ultrapure water and stored at 4°C for future use.

[0064] (2) Preparation of substrate materials

[0065] Ti-MOF / KB dispersion: 3.1 mmol 2-aminoterephthalic acid (NH2-BDC) was added into 15 mL N,N-dimethylformamide (DMF)-methanol mixed solvent (V DMF :V 甲醇＝ 9:1), then add 2.0mmol tetrabutyl titanate and 10mg Ketjen black (KB), ultrasonicate at room temperature for 10min, transfer the above solution to a reactor and react at 150℃ for 24h, cool, centrifuge at 10000rpm for 5min, wash with DMF and methanol and dry at 60℃ to obtain dark green Ti-MOF / KB nanomaterials; take 1mg of the above material and dissolve it in 1mL ultrapure water to obtain Ti-MOF / KB dispersion.

[0066] (3) Construction of an electrochemical immunosensor for detecting zearalenone:

[0067] 1) treating the capture antibody Ab1 with 0.1M PBS (pH=7) buffer at room temperature, treating the zearalenone antigen with ultrapure water, and storing them for later use;

[0068] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2=3:1, v / v) for 30 min and then rinse with ultrapure water for later use;

[0069] 3) The electrode obtained in step 2) was polished to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders, and then the electrode was ultrasonically treated in the order of ultrapure water, anhydrous ethanol, and ultrapure water, and dried for later use;

[0070] 4) electrochemically activating the electrode obtained in step 3) in 0.5M H2SO4, then rinsing with ultrapure water and drying;

[0071] 5) 10 μL of Ti-MOF / KB dispersion was added dropwise onto the surface of the cleaned glassy carbon electrode in step 4), and after drying at room temperature, 10 μL of AuNPs was added dropwise onto the surface of the electrode and dried at room temperature;

[0072] 6) Add 10 μL of Ab1 dropwise onto the electrode prepared in step 5) and incubate overnight at 4°C in a refrigerator for 12 h;

[0073] 7) After the electrode obtained in step 6) is rinsed with DEPC water, 5 μL of 1% bovine serum albumin (BSA) solution is added and incubated at 4°C for 30 min;

[0074] 8) After the electrode obtained in step 7) is rinsed with DEPC water, different concentrations of zearalenone are added dropwise to the electrode surface and incubated at 4° C. for 1 h;

[0075] 9) After the electrode obtained in step 8) is rinsed with DEPC water, 10 μL of PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution is added dropwise and incubated at room temperature for 4 h to obtain an electrochemical immunosensor for the detection of zearalenone.

[0076] The electrochemical immunosensor constructed in Example 1 was used to detect zearalenone in the following steps:

[0077] 1. Draw a standard curve

[0078] 1) The modified electrode of step 4) to step 9) in step (3) of Example 1 was placed in a 0.1M PBS (pH=7.0) solution for characterization, and the DPV response values ​​of different concentrations of zearalenone were measured. A working curve was drawn based on the linear relationship between the obtained current value and the logarithmic value of different zearalenone concentrations;

[0079] The detection results of the sensor of the present invention for different concentrations of zearalenone are as follows: Figure 1 shown. Figure 1 The calibration curve of the logarithmic value of zearalenone at different concentrations and the DPV current response value of the sensor is shown in Figure 3. The test results show that the logarithmic value of zearalenone concentration and the DPV current response value of the sensor have a good linear relationship in the concentration range of 10fg / mL-100ng / mL, the linear correlation coefficient is 0.9982, and the detection limit is 0.49fg / mL.

[0080] 2) The modified electrodes of step 4) to step 9) in step (3) of Example 1 were placed in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for CV. The current response signal was measured. The results are as follows: Figure 4 Shown: (a) bare glassy carbon electrode; (b) adding Ti-MOF / KB composite material; (d) adding AuNPs; (d) incubating Ab1; (e) adding BSA; (f) incubating the target.

[0081] 2. Sensor stability test:

[0082] The sensor prepared in Example 1 was scanned continuously for 25 cycles using differential pulse voltammetry (DPV). The results showed that the peak current value of the differential pulse voltammetry (DPV) curve obtained after 25 cycles of continuous scanning was 96.46% of the initial current, indicating that the sensor had good short-term stability. The prepared sensor was stored at 4°C for 14 days. The results showed that the current after 14 days of storage was 86.50% of the initial current, indicating that the sensor had good long-term stability.

[0083] 3. Sensor reproducibility test:

[0084] The sensor prepared by incubating the same concentration of zearalenone (ZEA) (1 pg / mL) with five different glassy carbon electrodes in Example 1 was measured (as shown in FIG. Figure 3 The relative standard deviation (RSD) was 3.04%, indicating that the sensor had good reproducibility.

Claims

1. An electrochemical immunosensor for detecting zearalenone ZEA, characterized in that: The method for constructing an electrochemical immunosensor for detecting zearalenone ZEA is as follows: adding a Ti-MOF / KB dispersion to a clean glassy carbon electrode surface, drying at room temperature, then adding an AuNPs solution to the electrode surface, drying at room temperature, adding Ab1 to the electrode, and incubating it in a refrigerator at 4-5°C overnight for 12-13 hours; rinsing it with DEPC water, adding a 1% bovine serum albumin BSA solution, and incubating it at 4-5°C for 30-35 minutes; finally, rinsing the obtained electrode with DEPC water, adding a PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution, and incubating it at room temperature for 4-5 hours to obtain an electrochemical immunosensor for detecting zearalenone ZEA; the preparation of the PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution is as follows: adding the zearalenone polyclonal antibody Ab2 to the prepared PDDA-NG / HNT / TB-AuNPs dispersion, stirring it in an ice bath for 12-13 hours. h, centrifuged, washed with water, and the precipitate was redispersed in ultrapure water to obtain a PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution; the preparation method of the PDDA-NG / HNT / TB-AuNPs dispersion is as follows: the prepared AuNPs are added to the prepared PDDA-NG / HNT / TB dispersion, stirred for 4-5h, centrifuged, washed, and the precipitate is dispersed in ultrapure water to obtain a PDDA-NG / HNT / TB-AuNPs dispersion; the preparation method of the PDDA-NG / HNT / TB dispersion is as follows: toluidine blue TB is added to the prepared PDDA-NG / HNT dispersion and stirred for 12-13 h, centrifuge, wash, and disperse the precipitate in ultrapure water to obtain a PDDA-NG / HNT / TB dispersion; the preparation method of the PDDA-NG / HNT dispersion is as follows: dissolve nitrogen-doped graphene NG in N,N-dimethylformamide, add polydimethylammonium chloride PDDA and stir for 2-3h, take the solution and add halloysite nanotubes HNT and stir for 3h to obtain a PDDA-NG / HNT dispersion; the preparation method of the Ti-MOF / KB dispersion is as follows: add 2-aminoterephthalic acid to a N,N-dimethylformamide-methanol mixed solvent and then add tetrabutyl titanate and Ketjen black KB, after ultrasonication at room temperature for 10-15 min, transfer the solution to a reactor and react at 150-160 ° C for 20-24 h, cool, centrifuge at 10000 rpm for 5-6 min, wash with DMF and methanol, and heat at 60-70 The obtained material was dried in an oven at ℃ to obtain a dark green Ti-MOF / KB nanomaterial; and then dissolved in ultrapure water to obtain a Ti-MOF / KB dispersion.

2. The electrochemical immunosensor for detecting zearalenone ZEA according to claim 1, characterized in that: The preparation method of the AuNPs is as follows: adding 1% HAuCl4 solution to ultrapure water and boiling, then adding 1% trisodium citrate solution and continuing to boil for 15 minutes, cooling and restoring the volume to the original volume with ultrapure water to obtain a transparent wine red solution, which is the nano-gold AuNPs.

3. An electrochemical immunosensor for detecting zearalenone, characterized in that: The method for constructing an electrochemical immunosensor for zearalenone detection comprises the following steps: (1) Preparation of signal probe: 1) PDDA-NG / HNT: Dissolve 10 mg of nitrogen-doped graphene NG in 10 mL of N,N-dimethylformamide, add 4 mL of polydimethylammonium chloride PDDA and stir for 2 h, take 2 mL of the solution and add 1 mg, 2 mg, 3 mg, 4 mg, and 5 mg of halloysite nanotubes HNT respectively and stir for 3 h to obtain PDDA-NG / HNT dispersion; 2) PDDA-NG / HNT / TB: Take 1 mL of the prepared PDDA-NG / HNT dispersion and add 1 mL of 1 mg / mL toluidine blue TB, stir for 12 h, centrifuge, wash with ultrapure water, and then disperse the black precipitate in 1 mL of ultrapure water to obtain a PDDA-NG / HNT / TB dispersion; 3) AuNPs: Add 1 mL of 1% HAuCl4 solution to 100 mL of ultrapure water and boil, then quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 minutes. After cooling, restore the volume to the original volume with ultrapure water to obtain a transparent wine-red solution, which is the nano-gold AuNPs. 4) PDDA-NG / HNT / TB-AuNPs: 500 μL of the prepared AuNPs was added to 1 mL of the prepared PDDA-NG / HNT dispersion material, stirred for 4 h, centrifuged, washed with ultrapure water, and the black precipitate was dispersed in 1 mL of ultrapure water to obtain a PDDA-NG / HNT / TB-AuNPs dispersion; 5) PDDA-NG / HNT / TB-AuNPs / Ab2: Add 200 μL of 10 μg / mL Ab2 to 1 mL of the prepared PDDA-NG / HNT / TB-AuNPs dispersion, stir in an ice bath for 12 h, centrifuge, wash with water, and redisperse the precipitate in 1 mL of ultrapure water to obtain the PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution; (2) Preparation of substrate materials Ti-MOF / KB dispersion: 3.1 mmol 2-aminoterephthalic acid was added to 15 mL of N,N-dimethylformamide-methanol mixed solvent with a volume ratio of 9:1, and then 2.0 mmol tetrabutyl titanate and 10 mg Ketjen black KB were added. Ultrasonication was performed at room temperature for 10 min, and the mixture was transferred to a reactor and reacted at 150 °C for 24 h. The mixture was cooled, centrifuged at 10,000 rpm for 5 min, washed with DMF and methanol, and dried in an oven at 60 °C to obtain dark green Ti-MOF / KB nanomaterials. 1 mg of Ti-MOF / KB nanomaterials was dissolved in 1 mL of ultrapure water to obtain Ti-MOF / KB dispersion. (3) Construction of an electrochemical immunosensor for detecting zearalenone: 1) Treat the capture antibody Ab1 and zearalenone antigen with 0.1M pH=7 PBS buffer at room temperature and store for later use; 2) Soak the glassy carbon electrode in piranha wash solution (98% H2SO4 / 30% H2O2 in a volume ratio of 3:1) for 30 min, then rinse with ultrapure water for later use; 3) The electrode obtained in step 2) was polished to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders, and then the electrode was ultrasonically treated in the order of ultrapure water, anhydrous ethanol, and ultrapure water, and dried for later use; 4) The electrode obtained in step 3) was electrochemically activated in 0.5 M H2SO4, then rinsed with ultrapure water and dried; 5) Add 10 μL of Ti-MOF / KB dispersion onto the cleaned glassy carbon electrode surface in step 4) and dry at room temperature. Then, add 10 μL of AuNPs onto the electrode surface and dry at room temperature. 6) Add 10 μL of Ab1 prepared in step 1) onto the electrode prepared in step 5) and incubate overnight in a refrigerator at 4°C for 12 hours; 7) Rinse the electrode obtained in step 6) with DEPC water, add 5 μL of 1% bovine serum albumin (BSA) solution and incubate at 4 °C for 30 min; 8) After the electrode obtained in step 7) is rinsed with DEPC water, different concentrations of zearalenone are added dropwise to the electrode surface and incubated at 4°C for 1 hour; 9) After rinsing the electrode obtained in step 8) with DEPC water, 10 μL of PDDA-NG / HNT / TB-AuNPs / Ab2 signal probe solution was added and incubated at room temperature for 4 h to obtain an electrochemical immunosensor for the detection of zearalenone.

4. A method for detecting zearalenone using the electrochemical immunosensor according to any one of claims 1 to 3, characterized in that: The steps include: 1) adding different concentrations of the target substance zearalenone to the electrode of the sensor; 2) Place the electrode in 0.1 M PBS solution with pH = 7.0 for characterization and measure its electrochemical signal; 3) drawing a working curve based on the linear relationship between the current value obtained in step 2) and the logarithmic value of the zearalenone concentration; 4) The sample to be tested is detected by the sensor, and the obtained current value is calculated through the working curve prepared in step 3) to obtain the concentration of zearalenone in the sample to be tested.

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