Preparation method of enzyme biosensor for simultaneously detecting multiple mycotoxins, sensor and application

Abstract

The invention discloses a preparation method of an enzyme biosensor for simultaneously detecting multiple mycotoxins, the sensor and application. According to the method, the carbon nano tube-nano gold laccase electrochemical biosensor is prepared through a layer-by-layer dispensing method, the preparation process is simple and rapid, operation is easy, the cost is low, a carbon nano tube modified electrode has reusability, the catalytic activity and stability of an enzyme modified by a nano material are remarkably improved, and the electrochemical biosensor has good application prospects. The obtained biosensor can perform high-flux simultaneous detection on mycotoxins such as aflatoxin B1, zearalenone and the like, is high in detection sensitivity, good in selectivity and small in reagent consumption, has the detection limit of the aflatoxin B1 of 0.012 mu mol / L and the detection range of 0.05-100 mu mol / L, has the detection limit of the zearalenone of 0.007 mu mol / L and the detection range of 0.01-120 mu mol / L, has the low detection limit and the wide detection range, and can be widely applied to detection of mycotoxins such as aflatoxin B1, zearalenone and the like. The problems of low flux, complex electrode preparation and poor stability and sensitivity of a traditional electrochemical detection mode are solved, and the method can be widely applied to rapid detection under the condition of co-pollution of multiple fungimycin in cereals.

Description

Technical Field

[0001] The present invention belongs to the field of grain detection and electrochemical sensing technology, and relates to an enzyme biosensor and a preparation method and application thereof, and specifically relates to an electrochemical enzyme biosensor for simultaneously detecting multiple fungal toxins and a preparation method and application thereof. Background Art

[0002] Mycotoxins are natural toxins produced by certain molds (fungi). They usually grow in crops and foods such as grains, nuts, and spices under warm and humid conditions. They may breed inside or on the surface of crops before and after harvest and during storage. Mycotoxins can have serious adverse effects on human and animal health. There are currently hundreds of known mycotoxins, the most common of which include aflatoxin, ochratoxin A, patulin, fumonisin, zearalenone, and nivalenol / deoxynivalenol. When crops are infected by mold before or after harvest, mycotoxins will enter the food chain. People may be directly exposed to mycotoxins by ingesting infected food, or indirectly exposed to mycotoxins by feeding contaminated feed to livestock, especially through animal milk, which may affect their health. Therefore, it is crucial to detect mycotoxins in crops such as grains or other foods and establish effective means of monitoring mycotoxins contamination.

[0003] In recent years, there have been more and more reports on the simultaneous contamination of multiple mycotoxins in crops or food and the synergistic toxicological effects of multiple toxins. The synergistic effects between mycotoxins can make the toxicity far greater than that produced by a single toxin, making the importance of simultaneous monitoring and control of multiple toxins increasingly prominent. Currently, the commonly used detection methods mainly include: thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC) combined with fluorescence detector (FLD) or mass spectrometry (MS), immunoassay, etc. Among them, the TLC method is simple to operate and suitable for the initial screening of a large number of samples, but the results are easily affected by subjectivity and are not suitable for quantitative analysis; the GC method is only suitable for the detection of vaporizable mycotoxins, which has great limitations; although the HPLC method combined with FLD or MS can accurately analyze the qualitative and quantitative analysis of mycotoxins, the detection equipment is large and the sample purification degree is strictly required, the detection cost is high and the detection efficiency is low; the immunoassay method is suitable for the needs of rapid on-site detection of large quantities of samples, and has the advantages of simple operation, high sensitivity, strong specificity and low cost, but the detection results are interfered by false negatives and false positives, and the accuracy is insufficient.

[0004] To solve the above technical problems, the industry has developed electrochemical detection methods, which have the advantages of high detection accuracy, low sample requirements, and low cost. However, they generally need to be combined with immunological elements for detection. The preparation and preservation of immunological elements have high requirements, and their detection mechanism is relatively cumbersome in terms of the generation of the analyte identification machine signal. Most importantly, most of them can only detect a single mycotoxin, and cannot perform high-throughput identification of complex contaminated mycotoxins, making it difficult to meet the detection needs when multiple mycotoxins exist at the same time. In addition, traditional electrochemical detection methods also have technical problems such as low sensitivity, narrow detection range, and complex electrode preparation.

[0005] In view of this, it is necessary to further improve the electrochemical mycotoxin detection device and detection method in the prior art. Summary of the invention

[0006] To this end, the technical problem to be solved by the present invention is that the existing electrochemical detection method of fungal toxins can only realize the detection of a single fungal toxin, with low flux, insufficient sensitivity, narrow detection range, and complex electrode preparation. Therefore, an electrochemical enzyme biosensor that can simultaneously detect two fungal toxins, with high throughput, high sensitivity, wide detection range, good stability of reaction elements, and simple electrode preparation is proposed, as well as a preparation method and application thereof.

[0007] In order to solve the above technical problems, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides a method for preparing an enzyme biosensor for simultaneously detecting multiple fungal toxins, comprising the following steps:

[0009] S1, dispersing the carboxylated carbon nanotubes in an organic solvent at a volume ratio of 1:1-1:3, and mixing them evenly to obtain a carboxylated carbon nanotube dispersion with a concentration of 0.3-1 mg / mL;

[0010] S2, coating the carboxylated carbon nanotube dispersion onto the pretreated surface of the bare gold electrode, and drying to obtain a carbon nanotube modified electrode;

[0011] S3, placing the carbon nanotube modified electrode in a HAuCl solution with a concentration of 0.1-1 mmol / L 4 In the solution, cyclic voltammetry scanning is performed to in-situ deposit nano-gold on the surface of the carbon nanotube modified electrode to obtain a carbon nanotube-nano-gold electrode;

[0012] S4. Apply a laccase solution with a concentration of 5-25 mg / mL to the surface of the carbon nanotube-nanogold electrode. After drying, drop perfluorosulfonic acid with a mass concentration of 0.1-1% onto the surface of the carbon nanotube-nanogold electrode coated with laccase to obtain a carbon nanotube-nanogold laccase electrochemical biosensor.

[0013] Preferably, in step S2, the preprocessing comprises the following steps:

[0014] S21, soak the bare gold electrode in piranha solution for 2-3 minutes, clean it, and polish it;

[0015] S22, placing the polished gold electrode in a first electrolyte for activation treatment, rinsing and drying for later use, wherein the first electrolyte is obtained by mixing potassium chloride, potassium ferrocyanide and potassium ferrocyanide solutions.

[0016] Preferably, in step S3, during the cyclic voltammetry scanning process, the potential interval is -0.2-1.0 V, the scanning speed is 50-100 mV / s, and the number of scanning cycles is 1-10 cycles.

[0017] Preferably, the solvent of the laccase solution is a PBS buffer with a concentration of 0.001-0.1 mol / L, and the pH value of the PBS buffer is 4-7.

[0018] Preferably, in step S2, the drying temperature is 50-70°C, the drying time is 4-6h, and the amount of the carboxylated carbon nanotubes coated on the pretreated surface of the bare gold electrode is 5-15 μL.

[0019] Preferably, in step S1, the organic solvent is N,N-dimethylformamide solution, and after the carboxylated carbon nanotubes are dispersed in the organic solvent, they are ultrasonically treated for 10-30 minutes and mixed evenly.

[0020] The second aspect of the present invention provides an enzyme biosensor, which is prepared by the preparation method.

[0021] Preferably, the method further comprises a second electrolyte, wherein the second electrolyte is a PBS buffer solution with a concentration of 0.001-0.1 mol / L, and the pH value of the PBS buffer solution is 4-7.

[0022] The third aspect of the present invention provides an application of the enzyme biosensor in detecting mycotoxins, wherein the mycotoxins include aflatoxins and zearalenone.

[0023] Preferably, when the electrochemical workstation detects mycotoxins, the sample to be tested is placed in the electrochemical workstation, a square wave voltammetry scan is performed at a scanning voltage of 0.2-1.2 V, and a quantitative analysis is performed based on substance concentration and peak current data.

[0024] The above technical solution of the present invention has the following advantages compared with the prior art:

[0025] The invention provides a method for preparing an enzyme biosensor for detecting multiple fungal toxins at the same time, comprising the following steps: S1, dispersing carboxylated carbon nanotubes in an organic solvent at a volume ratio of 1:1-1:3, and mixing them evenly to obtain a carboxylated carbon nanotube dispersion with a concentration of 0.3-1 mgm / L; S2, coating the carboxylated carbon nanotube dispersion on a pretreated bare gold electrode surface, and drying to obtain a carbon nanotube modified electrode; S3, placing the carbon nanotube modified electrode in a HAuCl4 solution with a concentration of 0.1-1 mmol / L, performing cyclic voltammetry scanning, and in-situ depositing nanogold on the surface of the carbon nanotube modified electrode to obtain a carbon nanotube-nanogold electrode; S4, coating a laccase solution with a concentration of 5-25 mg / mL on the surface of the carbon nanotube-nanogold electrode, and after drying, drop-coating a perfluorosulfonic acid film with a mass concentration of 0.1-1% on the surface of the carbon nanotube-nanogold electrode coated with laccase to obtain a carbon nanotube-nanogold laccase electrochemical biosensor. The method prepares a carbon nanotube-nanogold laccase electrochemical biosensor by a layer-by-layer drop coating method. The electrode preparation process is simple, rapid, easy to operate, and low in cost. The prepared carbon nanotube modified electrode is reusable. The catalytic activity and stability of the enzyme are significantly improved after modification with nanomaterials. The obtained biosensor can simultaneously detect aflatoxin B1, zearalenone and other fungal toxins with high throughput, and has high detection sensitivity, good selectivity, and low reagent consumption. The detection limit of aflatoxin B1 is 0.012 μmol / L, and the detection range is 0.05-100 μmol / L. The detection limit of zearalenone is 0.007 μmol / L, and the detection range is 0.01-120 μmol / L. It has a low detection limit and a wide detection range, which solves the problems of low flux, complex electrode preparation, poor stability and sensitivity of traditional electrochemical detection methods, and can be widely used in the rapid detection of multiple fungal toxins co-contaminated in cereals. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0027] Figure 1 is a cyclic voltammetry curve diagram of the electrode obtained at different stages by the preparation method provided in Example 1 of the present invention;

[0028] Figure 2 is an impedance analysis curve diagram of the electrode obtained at different stages by the preparation method provided in Example 1 of the present invention;

[0029] Figure 3 It is a SWV test curve diagram of the carbon nanotube-nanogold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 of the present invention in PBS buffer at different pH values;

[0030] Figure 4 It is a SWV test curve diagram of the carbon nanotube-nano-gold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 of the present invention in PBS buffer solutions of different concentrations;

[0031] Figure 5 This is a cyclic voltammetry test curve of the carbon nanotube-nano-gold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 of the present invention on a sample containing aflatoxin B1;

[0032] Figure 6 This is a cyclic voltammetric test curve of the carbon nanotube-nano-gold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 of the present invention on a sample containing zearalenone;

[0033] Figure 7 It is a SWV test curve diagram of the carbon nanotube-nanogold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 of the present invention for samples containing aflatoxin B1 and zearalenone at different aflatoxin B1 concentrations;

[0034] Figure 8 It is a SWV test curve of the carbon nanotube-nanogold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 of the present invention for samples containing aflatoxin B1 and zearalenone at different zearalenone 1 concentrations. DETAILED DESCRIPTION

[0035] Example 1

[0036] This embodiment provides a method for preparing an enzyme biosensor for simultaneously detecting multiple fungal toxins, the method comprising the following steps:

[0037] S1. Disperse the carboxylated carbon nanotubes in an organic solvent of N,N-dimethylformamide (DMF) at a volume ratio of 1:1, and perform ultrasonication for 30 minutes until the mixture is uniformly mixed to form a carboxylated carbon nanotube dispersion having a concentration of 1 mg / mL, wherein the carboxylated carbon nanotubes are commercially available carboxylated carbon nanotube products.

[0038] S2. 15 μL of the carboxylated nanotube dispersion obtained in step S1 is applied to the pretreated surface of the bare gold electrode, and then the gold electrode coated with the carboxylated nanotube dispersion is placed in an oven and dried at 70° C. to obtain a carbon nanotube modified electrode.

[0039] In this embodiment, the bare gold electrode is pretreated by the following steps:

[0040] S21. Soak the bare gold electrode in the piranha solution for 3 minutes, take it out, place it in 99% anhydrous ethanol for ultrasonic cleaning for 2-3 minutes, and then place it in distilled water for ultrasonic cleaning for 2-3 minutes. Then, cover the bare gold electrode with a layer of aluminum powder (Al 2 O 3 ) suede cloth to polish the electrode surface to a mirror surface and then rinse it clean. The piranha solution is a mixed solution formed by mixing a 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:7.

[0041] S22. Place the polished gold electrode in a first electrolyte for activation treatment. After the activation treatment, take it out, rinse it, and blow it dry with nitrogen for later use. The electrolyte is a mixed aqueous solution of potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3.

[0042] S3, place the carbon nanotube modified electrode in a 1 mmol / L HAuCl 4 In the solution, cyclic voltammetry was performed. During the cyclic voltammetry scan, the potential range was -0.2-1V, the scan rate was 100mV / s, and the number of scans was 1 circle, thereby HAuCl 4 Reduction was performed to in-situ deposit gold nanoparticles on the surface of the carbon nanotube-modified electrode. After scanning, the electrode was cleaned with ethanol and redistilled water and dried at room temperature for 6 h to obtain a carbon nanotube-gold nanoparticle electrode.

[0043] S4. Take a laccase solution with a concentration of 25 mg / mL and drop it on the surface of the carbon nanotube-nanogold electrode. After drying at room temperature, a carbon nanotube-nanogold laccase modified electrode is obtained. 10 μL of a perfluorosulfonic acid (Nafion) solution with a mass concentration of 1% is drop-coated on the surface of the carbon nanotube-nanogold electrode coated with laccase to obtain a carbon nanotube-nanogold laccase electrochemical biosensor, wherein the solvent in the laccase solution is PBS buffer solution. In this embodiment, its concentration is 0.1 mol / L and the pH value is 7.

[0044] The present embodiment provides a method for preparing an enzyme biosensor for simultaneously detecting multiple fungal toxins. The carbon nanotube-nanogold laccase electrochemical biosensor is prepared by a layer-by-layer drop coating method. This method has the advantages of small reagent consumption and low preparation cost. The electrode preparation process is simple, fast, easy to operate and low in cost. The prepared carbon nanotube modified electrode is reusable. The catalytic activity and stability of the enzyme modified by carbon nanotubes and nanogold are significantly improved. The obtained biosensor can simultaneously detect aflatoxin B1, zearalenone and other fungal toxins with high throughput, and has high detection sensitivity, good selectivity and low reagent consumption. The detection limit of aflatoxin B1 is 0.012 μmol / L, and the detection range is 0.05-100 μmol / L. The detection limit of zearalenone is 0.007 μmol / L, and the detection range is 0.01-120 μmol / L. It has a low detection limit and a wide detection range, which solves the problems of low flux, complex electrode preparation, poor stability and sensitivity of traditional electrochemical detection methods, and can be widely used in rapid detection of multiple fungal toxins co-contaminated in complex food matrices or cereals.

[0045] This embodiment also provides an electrochemical enzyme biosensor, which is prepared by the above preparation method.

[0046] Furthermore, a second electrolyte is included. The enzyme biosensor and the second electrolyte can form an electrochemical workstation. In this embodiment, the second electrolyte uses a PBS buffer solution with a concentration of 0.01 mol / L and a pH of 4.5.

[0047] This embodiment also provides an application of the electrochemical enzyme biosensor prepared by the above preparation method in detecting fungal toxins, wherein the fungal toxins include at least aflatoxin B1 and zearalenone, that is, the electrochemical enzyme biosensor can be used for the simultaneous detection of multiple fungal toxins.

[0048] In this embodiment, the application of the electrochemical enzyme biosensor in the detection of mycotoxins specifically includes the following steps:

[0049] The sample to be tested is placed in the above-mentioned electrochemical workstation (i.e., the electrochemical workstation obtained by placing the electrochemical enzyme biosensor in PBS buffer), and a square wave voltammetric scan is performed at a scanning voltage of 0.2-1.2V. The control unit of the electrochemical workstation records the change in current-voltage to obtain a current-voltage curve (voltammetric curve). Quantitative analysis is performed based on the linear relationship between substance concentration and current. Specifically, the electrochemical peaks of about 0.5V and 0.8V represent the oxidation of aflatoxin B1 and zearalenone, respectively, thereby achieving the effect of detecting aflatoxin B1 and zearalenone in the sample to be tested.

[0050] Example 2

[0051] This embodiment provides a method for preparing an enzyme biosensor for simultaneously detecting multiple fungal toxins, the method comprising the following steps:

[0052] S1. Disperse the carboxylated carbon nanotubes in an organic solvent of N,N-dimethylformamide (DMF) at a volume ratio of 1:3, and perform ultrasonication for 10 min until the mixture is uniformly mixed to form a carboxylated carbon nanotube dispersion having a concentration of 0.3 mg / mL, wherein the carboxylated carbon nanotubes are commercially available carboxylated carbon nanotube products.

[0053] S2. 5 μL of the carboxylated nanotube dispersion obtained in step S1 is applied to the pretreated surface of the bare gold electrode, and then the gold electrode coated with the carboxylated nanotube dispersion is placed in an oven and dried at 50° C. to obtain a carbon nanotube modified electrode.

[0054] In this embodiment, the bare gold electrode is pretreated by the following steps:

[0055] S21. Soak the bare gold electrode in the piranha solution for 2 minutes, take it out, place it in 99% anhydrous ethanol for ultrasonic cleaning for 2-3 minutes, and then place it in distilled water for ultrasonic cleaning for 2-3 minutes. Then, cover the bare gold electrode with a layer of aluminum powder (Al 2 O 3 ) suede cloth to polish the electrode surface to a mirror surface and then rinse it clean. The piranha solution is a mixed solution formed by mixing a 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:7.

[0056] S22. Place the polished gold electrode in a first electrolyte for activation treatment. After the activation treatment, take it out, rinse it, and blow it dry with nitrogen for later use. The first electrolyte is a mixed aqueous solution of potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3.

[0057] S3, place the carbon nanotube modified electrode in a HAuCl solution with a concentration of 0.1 mmol / L 4 In the solution, cyclic voltammetry was performed. During the cyclic voltammetry scan, the potential range was -0.2-1V, the scan rate was 50mV / s, and the number of scans was 10, thereby HAuCl 4 Reduction was performed to in-situ deposit gold nanoparticles on the surface of the carbon nanotube-modified electrode. After scanning, the electrode was cleaned with ethanol and redistilled water and dried at room temperature for 4 h to obtain a carbon nanotube-gold nanoparticle electrode.

[0058] S4. Take a laccase solution with a concentration of 5 mg / mL and drop it on the surface of the carbon nanotube-nanogold electrode. After drying at room temperature, 4 μL of a perfluorosulfonic acid (Nafion) solution with a mass concentration of 0.1% is drop-coated on the surface of the carbon nanotube-nanogold electrode coated with laccase to obtain a carbon nanotube-nanogold laccase electrochemical biosensor, wherein the solvent in the laccase solution is PBS buffer solution. In this embodiment, its concentration is 0.001 mol / L and the pH value is 4.

[0059] This embodiment also provides an electrochemical enzyme biosensor, which is prepared by the above preparation method.

[0060] Furthermore, a second electrolyte is included. The enzyme biosensor and the second electrolyte can form an electrochemical workstation. In this embodiment, the second electrolyte uses a PBS buffer solution with a concentration of 0.001 mol / L and a pH of 4.

[0061] This embodiment also provides an application of the electrochemical enzyme biosensor prepared by the above preparation method in detecting fungal toxins, wherein the fungal toxins include at least aflatoxin B1 and zearalenone, that is, the electrochemical enzyme biosensor can be used for the simultaneous detection of multiple fungal toxins.

[0062] In this embodiment, the application of the electrochemical enzyme biosensor in the detection of mycotoxins specifically includes the following steps:

[0063] The sample to be tested is placed in the above-mentioned electrochemical workstation (i.e., the electrochemical workstation obtained by placing the electrochemical enzyme biosensor in PBS buffer), and a square wave voltammetric scan is performed at a scanning voltage of 0.2-1.2V. The control unit of the electrochemical workstation records the change in current-voltage to obtain a current-voltage curve (voltammetric curve). Quantitative analysis is performed based on the linear relationship between substance concentration and current. Specifically, the electrochemical peaks of about 0.5V and 0.8V represent the oxidation of aflatoxin B1 and zearalenone, respectively, thereby achieving the effect of detecting aflatoxin B1 and zearalenone in the sample to be tested.

[0064] Example 3

[0065] This embodiment provides a method for preparing an enzyme biosensor for simultaneously detecting multiple fungal toxins, the method comprising the following steps:

[0066] S1. Disperse the carboxylated carbon nanotubes in an organic solvent of N,N-dimethylformamide (DMF) at a volume ratio of 1:2, and perform ultrasonication for 20 minutes until the mixture is uniformly mixed to form a carboxylated carbon nanotube dispersion having a concentration of 0.6 mg / mL, wherein the carboxylated carbon nanotubes are commercially available carboxylated carbon nanotube products.

[0067] S2. Pipette 10 μL of the carboxylated nanotube dispersion obtained in step S1 and apply it to the pretreated surface of the bare gold electrode. Then place the gold electrode coated with the carboxylated nanotube dispersion in an oven and dry it at 60° C. to obtain a carbon nanotube modified electrode.

[0068] In this embodiment, the bare gold electrode is pretreated by the following steps:

[0069] S21. Soak the bare gold electrode in the piranha solution for 3 minutes, take it out, place it in 99% anhydrous ethanol for ultrasonic cleaning for 2-3 minutes, and then place it in distilled water for ultrasonic cleaning for 2-3 minutes. Then, cover the bare gold electrode with a layer of aluminum powder (Al 2 O 3 ) suede cloth to polish the electrode surface to a mirror surface and then rinse it clean. The piranha solution is a mixed solution formed by mixing a 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:7.

[0070] S22. Place the polished gold electrode in a first electrolyte for activation treatment. After the activation treatment, take it out, rinse it, and blow it dry with nitrogen for later use. The first electrolyte is a mixed aqueous solution of potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3.

[0071] S3, place the carbon nanotube modified electrode in a HAuCl solution with a concentration of 0.05 mmol / L 4 In the solution, cyclic voltammetry was performed. During the cyclic voltammetry scan, the potential range was -0.2-1V, the scan rate was 70mV / s, and the number of scans was 5, so that HAuCl 4 Reduction was performed to in-situ deposit gold nanoparticles on the surface of the carbon nanotube-modified electrode. After scanning, the electrode was cleaned with ethanol and redistilled water and dried at room temperature for 5 h to obtain a carbon nanotube-gold nanoparticle electrode.

[0072] S4. Take a laccase solution with a concentration of 15 mg / mL and drop it on the surface of the carbon nanotube-nanogold electrode. After drying at room temperature, 7 μL of a perfluorosulfonic acid (Nafion) solution with a mass concentration of 0.5% is drop-coated on the surface of the carbon nanotube-nanogold electrode coated with laccase to obtain a carbon nanotube-nanogold laccase electrochemical biosensor, wherein the solvent in the laccase solution is PBS buffer solution. In this embodiment, its concentration is 0.05 mol / L and the pH value is 5.

[0073] This embodiment also provides an electrochemical enzyme biosensor, which is prepared by the above preparation method.

[0074] Furthermore, a second electrolyte is included. The enzyme biosensor and the second electrolyte can form an electrochemical workstation. In this embodiment, the second electrolyte is a PBS buffer solution with a concentration of 0.1 mol / L and a pH of 7.

[0075] This embodiment also provides an application of the electrochemical enzyme biosensor prepared by the above preparation method in detecting fungal toxins, wherein the fungal toxins include at least aflatoxin B1 and zearalenone, that is, the electrochemical enzyme biosensor can be used for the simultaneous detection of multiple fungal toxins.

[0076] In this embodiment, the application of the electrochemical enzyme biosensor in the detection of fungal toxins specifically includes the following steps:

[0077] The sample to be tested is placed in the above-mentioned electrochemical workstation (i.e., the electrochemical workstation obtained by placing the electrochemical enzyme biosensor in PBS buffer), and a square wave voltammetric scan is performed at a scanning voltage of 0.2-1.2V. The control unit of the electrochemical workstation records the change in current-voltage to obtain a current-voltage curve (voltammetric curve). Quantitative analysis is performed based on the linear relationship between substance concentration and current. Specifically, the electrochemical peaks of about 0.5V and 0.8V represent the oxidation of aflatoxin B1 and zearalenone, respectively, thereby achieving the effect of detecting aflatoxin B1 and zearalenone in the sample to be tested.

[0078] Experimental example

[0079] 1. Electrode characterization

[0080] The electrochemical properties of the electrodes obtained at different stages by the preparation method provided in Example 1 were tested respectively. The test results are as follows: Figure 1-Figure 2 As shown, Figure 1 is the cyclic voltammetry curve of each electrode, Figure 1 Curve 1 is the cyclic voltammetry curve of the bare gold electrode, curve 2 is the cyclic voltammetry curve of the carbon nanotube modified electrode, curve 3 is the cyclic voltammetry curve of the carbon nanotube-nano-gold electrode, and curve 4 is the cyclic voltammetry curve of the carbon nanotube-nano-gold laccase modified electrode.

[0081] Figure 2 These are the impedance analysis curves of bare gold electrode, carbon nanotube modified electrode, carbon nanotube-nanogold electrode, and carbon nanotube-nanogold laccase modified electrode.

[0082] It can be seen from the test results that with the successful modification of carbon nanotubes and nanogold on the surface of the gold electrode, the peak signal gradually increases and the resistance gradually decreases due to the excellent conductivity and electron transmission ability of the modified materials. After the modification of laccase, the binding of enzyme protein substances increases the steric hindrance, resulting in a decrease in the current signal compared with the bare gold electrode, carbon nanotubes, and carbon nanotube-nanogold modified gold electrode, which proves that the laccase has been successfully modified.

[0083] 2. The SWV (square wave voltammetry) test graph of the carbon nanotube-nano-gold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 in PBS buffer (concentration of 0.01 M) at different pH values ​​was tested. The test results are as follows: Figure 3 shown.

[0084] It can be seen from the figure that different pH values ​​have an effect on the current response and peak potential, and the best current response is achieved at pH 4.5.

[0085] 3. The SWV (square wave voltammetry) test graph of the carbon nanotube-nano-gold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 in PBS buffer (pH 4.5) of different concentrations was tested. The test results are as follows: Figure 4 As shown, Figure 4 In the figure, curves 1 to 5 are test curves when the concentrations of PBS buffer are 0.1M, 0.001M, 0.005M, 0.01M, and 0.05M, respectively.

[0086] It can be seen from the figure that different PBS solution concentrations have an effect on the current response and peak potential. When the concentration of the PBS solution is 0.01M, it has the best current response.

[0087] 4. The carbon nanotube-nano-gold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 was tested for the results of cyclic voltammetry on samples containing aflatoxin B1 and samples containing zearalenone. The CV test results of the samples containing aflatoxin B1 at different scanning speeds of 20-200 mV / S were as follows: Figure 5 As shown in the figure, the CV test results of samples containing zearalenone are as follows: Figure 6 shown.

[0088] The test results show that the current response increases with the increase of scanning speed.

[0089] 5. Testing The carbon nanotube-nano-gold laccase electrochemical biosensor prepared by the preparation method provided in Example 1 was tested for SWV on a sample containing both aflatoxin B1 and zearalenone. The test curve is shown in the figure below. Figure 7-Figure 8 As shown, Figure 7It is the SWV curve under different aflatoxin B1 concentrations. Figure 8 It is the SWV curve diagram under different zearalenone concentrations.

[0090] It can be seen from the figure that as the concentration of aflatoxin B1 gradually increases in the range of 0.05-100 μmol / L, the peak current of the carbon nanotube-nano-gold laccase electrochemical biosensor shows a good linear relationship in the above concentration range, satisfying the following linear equation: I AFB1 =0.0305x+0.5125(R 2 =0.9982), the detection limit is 0.012 μmol / L, and it can be seen that the oxidation potential of aflatoxin B1 is about 0.48 V.

[0091] The results of zearalenone were similar. Figure 8 As shown in Figure 2, within the range of 0.01-120 μmol / L, the peak current increases with the increase of the concentration of zeaxanthin, and the regression equation is I ZEA =0.021x+0.7608(R 2 =0.9967), the detection limit is 0.007 μmol / L, it can be seen that the oxidation potential of zearalenone is about 0.8V.

[0092] 6. The SWV electrochemical analysis method combined with the standard addition method was used to simultaneously detect aflatoxin B1 (AFB1) and zearalenone (ZEN) in rice flour samples. In the rice flour samples, aflatoxin B1 and zearalenone were spiked at concentrations of 1 μmol / L, 10 μmol / L, and 100 μmol / L, respectively. The test results are shown in Table 1 below:

[0093] Table 1

[0094]

[0095] The test results show that after the two mycotoxin substances were spiked into the rice noodle sample, the recovery rates of the two mycotoxin substances were both above 90%, indicating that the electrochemical enzyme biosensor prepared by the method provided in the embodiment of the present application has good detection performance for the sample and can be applied to the detection of samples containing the above-mentioned mycotoxins.

[0096] In summary, the electrochemical enzyme biosensor prepared by the preparation method provided in the present application has a synergistic effect between carboxylated carbon nanotubes and nanogold, contains a large number of active sites for redox reactions, enhances the electron transfer effect of the gold electrode, reduces the electrochemical impedance, increases the electrochemical specific surface area, and also improves the catalytic activity and stability of the enzyme. The electrochemical biosensor modified with carboxylated carbon nanotubes, nanogold, laccase, and Nafion exhibits excellent electrochemical performance, and shows a good linear relationship in the detection of two fungal toxins in the concentration ranges of 0.05-100 μmol / L and 0.01-100 μmol / L, with detection limits of 0.012 μmol / L and 0.007 μmol / L, respectively. It can realize direct electrochemical simultaneous detection of aflatoxin B1 and zearalenone, and solves the technical problems existing in commonly used detection technologies and current electrochemical detection methods.

[0097] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing an enzyme biosensor for simultaneously detecting multiple fungal toxins, characterized in that: The steps include: S1, dispersing the carboxylated carbon nanotubes in an organic solvent at a volume ratio of 1:1-1:3, and mixing them evenly to obtain a carboxylated carbon nanotube dispersion with a concentration of 0.3-1 mg / mL; S2, coating the carboxylated carbon nanotube dispersion onto the pretreated surface of a bare gold electrode, and drying to obtain a carbon nanotube modified electrode; S3, placing the carbon nanotube modified electrode in a HAuCl4 solution with a concentration of 0.1-1 mmol / L, performing cyclic voltammetry scanning, and in-situ deposition of nanogold on the surface of the carbon nanotube modified electrode to obtain a carbon nanotube-nanogold electrode; S4. Apply a laccase solution with a concentration of 5-25 mg / mL to the surface of the carbon nanotube-nanogold electrode. After drying, drop perfluorosulfonic acid with a mass concentration of 0.1-1% onto the surface of the carbon nanotube-nanogold electrode coated with laccase to obtain a carbon nanotube-nanogold laccase electrochemical biosensor.

2. The method for preparing an enzyme biosensor for simultaneously detecting multiple fungal toxins according to claim 1, characterized in that: In step S2, the preprocessing includes the following steps: S21, soak the bare gold electrode in piranha solution for 2-3 minutes, clean it, and polish it; S22, placing the polished gold electrode in a first electrolyte for activation treatment, rinsing and drying for later use, wherein the first electrolyte is obtained by mixing potassium chloride, potassium ferrocyanide and potassium ferrocyanide solutions.

3. The method for preparing an enzyme biosensor for simultaneously detecting multiple mycotoxins according to claim 1 or 2, characterized in that: In step S3, during the cyclic voltammetry scanning process, the potential interval is -0.2-1.0 V, the scanning speed is 50-100 mV / s, and the number of scanning circles is 1-10 circles.

4. The method for preparing an enzyme biosensor for simultaneously detecting multiple fungal toxins according to claim 3, characterized in that: The solvent of the laccase solution is a PBS buffer solution with a concentration of 0.001-0.1 mol / L, and the pH value of the PBS buffer solution is 4-7.

5. The method for preparing an enzyme biosensor for simultaneously detecting multiple fungal toxins according to claim 4, characterized in that: In step S2, the drying temperature is 50-70°C, the drying time is 4-6 hours, and the amount of the carboxylated carbon nanotubes coated on the pretreated surface of the bare gold electrode is 5-15 μL.

6. The method for preparing an enzyme biosensor for simultaneously detecting multiple mycotoxins according to claim 5, characterized in that: In step S1, the organic solvent is N,N-dimethylformamide solution, the carboxylated carbon nanotubes are dispersed in the organic solvent, and then ultrasonicated for 10-30 minutes to mix evenly.

7. An enzyme biosensor, characterized in that The enzyme biosensor is prepared by the preparation method according to any one of claims 1 to 6.

8. The enzyme biosensor according to claim 7, characterized in that It also includes a second electrolyte, which is a PBS buffer solution with a concentration of 0.001-0.1 mol / L, and a pH value of the PBS buffer solution is 4-7.

9. Use of the enzyme biosensor according to claim 7 or 8 in detecting mycotoxins, wherein the mycotoxins include aflatoxins and zearalenone.

10. The use according to claim 9, characterized in that: When the electrochemical workstation detects mycotoxins, the sample to be tested is placed in the electrochemical workstation, a square wave voltammetry scan is performed at a scanning voltage of 0.2-1.2 V, and a quantitative analysis is performed based on substance concentration and peak current data.

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