Preparation method of composite electrochemical probe for detecting vomitoxin concentration and detection method of vomitoxin concentration

Abstract

The present invention discloses a preparation method of a composite electrochemical probe for detecting the concentration of vomitoxin and a detection method of the concentration of vomitoxin, comprising the following preparation steps: mixing zinc nitrate, Prussian blue and 2-methylimidazole evenly, filtering after centrifugation, obtaining a first precipitate, mixing the first precipitate with a Tris-HCl buffer solution, adding a hydrochloric acid dopamine solution, stirring evenly, filtering after centrifugation, obtaining a second precipitate, dispersing the second precipitate in a phosphate buffer, and obtaining a PB@ZIF-8 / PDA solution; adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to IgG, mixing evenly, and obtaining activated IgG; adding the activated IgG to the PB@ZIF-8 / PDA solution, incubating, shaking, centrifuging and filtering, obtaining a third precipitate, washing the third precipitate, and obtaining a PB@ZIF-8 / PDA / IgG signal probe. The composite electrochemical probe for detecting the concentration of vomitoxin prepared by the preparation method provided by the present invention can detect the concentration of vomitoxin simply, quickly and stably.

Description

Technical Field

[0001] The present invention relates to the technical field of vomitoxin detection, and in particular to a method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin and a method for detecting the concentration of vomitoxin. Background Art

[0002] Deoxynivalenol (DON) is a metabolite produced by Fusarium graminearum, Fusarium oxysporum, Fusarium moniliforme, Fusarium pseudocladosporum, Fusarium pink, Fusarium nivalis, etc. It has stable chemical properties and has stable properties such as heat resistance, pressure resistance, acid resistance, and storage resistance. It is difficult to be destroyed during processing. It has always been an important hidden danger in the process of grain production and processing, and is listed as a Class 3 carcinogen by the International Agency for Research on Cancer. DON is widely present in food and the environment, mainly contaminating grain crops such as wheat, barley, and corn. Humans and animals will have a wide range of toxic effects after accidentally eating food contaminated by DON. DON can contaminate food during the growth, harvesting, storage, transportation, processing, and circulation of grain, posing a huge threat to food safety and human health. In order to ensure food safety and protect the rights and interests of consumers, accurate detection of DON content in grain is crucial to determine the degree of DON contamination. Therefore, it is urgent to develop a low-cost, highly sensitive, accurate and rapid DON detection method.

[0003] Traditional detection methods for DON mainly include thin layer chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry, immunoaffinity column purification-high performance liquid chromatography, etc. These methods have defects such as complex sample pre-processing, expensive equipment, complex detection procedures, and the need for professional testing personnel. They are not suitable for the detection of large quantities of samples, and it is difficult to meet the needs of rapid detection results in the grassroots supervision process, and it is not convenient to promote and apply them. Summary of the invention

[0004] The main purpose of the present invention is to propose a method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin and a method for detecting the concentration of vomitoxin, aiming to provide a composite electrochemical probe for detecting the concentration of vomitoxin, which can detect the concentration of vomitoxin simply, quickly and stably.

[0005] To achieve the above object, the present invention provides a method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin, comprising the following preparation steps:

[0006] Mix zinc nitrate, Prussian blue and 2-methylimidazole evenly, centrifuge and filter to obtain a first precipitate, mix the first precipitate with a Tris-HCl buffer solution, add a dopamine hydrochloride solution, stir evenly, centrifuge and filter to obtain a second precipitate, disperse the second precipitate in a phosphate buffer to obtain a PB@ZIF-8 / PDA solution;

[0007] Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to IgG and mixing well to obtain activated IgG;

[0008] Adding the activated IgG to the PB@ZIF-8 / PDA solution, incubating, shaking, centrifuging, and filtering to obtain a third precipitate, washing the third precipitate to obtain a PB@ZIF-8 / PDA / IgG signal probe;

[0009] Grind and clean the electrode, then dry it with protective gas to obtain a clean electrode;

[0010] Sequentially dropping 3,3'-disulfide and DON-bovine serum albumin onto the clean electrode to obtain a modified electrode, placing the modified electrode in a BSA solution, and incubating to obtain a DON-BSA / DTSSP / Au electrode;

[0011] The DON-BSA / DTSSP / Au electrode was modified with a mixture of DON antibodies and DON antigens containing different concentration gradients, and then the PB@ZIF-8 / PDA / IgG signal probe was added dropwise to obtain a composite electrochemical probe for detecting DON.

[0012] Optionally, zinc nitrate, Prussian blue and 2-methylimidazole are mixed uniformly, centrifuged and filtered to obtain a first precipitate, the first precipitate is mixed with a Tris-HCl buffer solution, a dopamine hydrochloride solution is added, the mixture is stirred uniformly, centrifuged and filtered to obtain a second precipitate, and the second precipitate is dispersed in a phosphate buffer to obtain a PB@ZIF-8 / PDA solution.

[0013] The volume ratio of the zinc nitrate, the Prussian blue and the 2-methylimidazole is 1:(0.8-1.2):(0.8-1.2); and / or,

[0014] The volume ratio of the Tris-HCl buffer solution to the dopamine hydrochloride solution is 1:(0.8-1.2); and / or,

[0015] The concentration of the zinc nitrate is 0.1-0.3 mol / L; and / or,

[0016] The concentration of the Prussian blue is 1.5 to 2.5 mmol / L; and / or,

[0017] The concentration of the 2-methylimidazole is 0.3-0.5 mol / L; and / or,

[0018] The concentration of the Tris-HCl buffer solution is 20-25 mmol / L; and / or,

[0019] The concentration of the dopamine hydrochloride solution is 0.3-0.5 mg / mL.

[0020] Optionally, in the step of adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to IgG and mixing them evenly to obtain activated IgG,

[0021] The volume ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the N-hydroxysuccinimide and the IgG is 1:(0.8-1.2):(20-30); and / or,

[0022] The concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.1 to 0.3 mmol / L; and / or,

[0023] The concentration of the N-hydroxysuccinimide is 0.2-0.5 mmol / L; and / or,

[0024] The concentration of the IgG is 0.3-0.5 mg / mL.

[0025] Optionally, the steps of adding the activated IgG to the PB@ZIF-8 / PDA solution, incubating, shaking, centrifuging, and filtering to obtain a third precipitate, washing the third precipitate, and obtaining the PB@ZIF-8 / PDA / IgG signal probe include:

[0026] The activated IgG is added to the PB@ZIF-8 / PDA solution, incubated for 30 to 40 minutes, and then shaken for 8 to 12 hours at 2 to 5°C. After centrifugation, a third precipitate is obtained, and the third precipitate is washed 2 to 3 times with a phosphate buffer solution. Then, bovine serum albumin is added, and the solution is incubated at 35 to 37°C for 0.8 to 1.2 hours to obtain a fourth precipitate. After washing the fourth precipitate 2 to 3 times with a phosphate buffer solution, the fourth precipitate is dispersed in a phosphate buffer solution.

[0027] Optionally, the concentration of the bovine serum albumin is 1.0-1.2%; and / or,

[0028] The concentration of the phosphate buffer is 8 to 10 mmol / L; and / or,

[0029] The pH value of the phosphate buffer is 7.2-7.5.

[0030] Optionally, the steps of cleaning the electrode and then drying the electrode with a protective gas to obtain a clean electrode include:

[0031] The electrode is polished in alumina powder, washed with deionized water, anhydrous ethanol, and deionized water for 5 to 10 minutes, and then washed with a mixture of hydrogen peroxide and concentrated sulfuric acid for 2 to 3 times to obtain an intermediate product electrode. The intermediate product electrode is placed in a sulfuric acid solution, subjected to constant potential and CV treatments in turn, taken out, washed, and dried in an inert environment to obtain a clean electrode.

[0032] Optionally, the diameter of the aluminum oxide powder is 0.3 μm and / or 0.5 μm; and / or,

[0033] In the mixed solution, the volume ratio of the hydrogen peroxide to the concentrated sulfuric acid is 1:(2.5-3.5); and / or,

[0034] The concentration of the sulfuric acid solution is 0.3-0.5 mol / L; and / or,

[0035] The protective gas includes nitrogen.

[0036] Optionally, the steps of sequentially dropping 3,3'-disulfide and DON-bovine serum albumin onto the clean electrode to obtain a modified electrode, placing the modified electrode in a BSA solution, and incubating to obtain a DON-BSA / DTSSP / Au electrode include:

[0037] 3,3'-disulfide was dropped onto the surface of the clean electrode, and the mixture was incubated at 25-30°C for 0.5-1.5h. The surface of the clean electrode was washed with a PBST solution. Then, vomitoxin-bovine serum albumin was dropped onto the surface of the clean electrode, and the mixture was incubated at 35-37°C for 1.5-2.5h. The surface of the clean electrode was washed with a PBST solution to obtain a modified electrode. The modified electrode was placed in a BSA solution and incubated for 0.5-1.5h to obtain a DON-BSA / DTSSP / Au electrode.

[0038] Optionally, the volume ratio of the 3,3'-disulfide to the DON-BSA is 1:(0.8-1.2); and / or,

[0039] The concentration of the 3,3'-disulfide is 1 to 3 mmol / L; and / or,

[0040] The concentration of the DON-BSA is 3-5 μg / mL; and / or,

[0041] The concentration of the BSA solution is 1-2%; and / or,

[0042] The concentration of the DON antibody is 4-5 μg / mL.

[0043] In addition, the present invention also proposes a method for detecting the concentration of vomitoxin, comprising the following detection steps:

[0044] Obtaining a working electrode, a reference electrode and an auxiliary electrode, wherein the working electrode comprises a composite electrochemical probe for detecting the concentration of vomitoxin;

[0045] The working electrode, the reference electrode and the auxiliary electrode are placed in a PBS buffer solution, and a standard concentration-current curve is obtained by differential pulse voltammetry;

[0046] The sample to be detected is modified on the working electrode, and then the working electrode, the reference electrode and the auxiliary electrode are placed in PBS buffer, and the current curve of the sample to be detected is obtained by differential pulse voltammetry. The current curve of the sample to be detected is compared with the standard concentration-current curve to obtain the vomitin concentration.

[0047] In the technical scheme of the present invention, Prussian blue has high catalytic activity and good electrochemical activity, which can make up for the deficiency of low detection efficiency, and Prussian blue is simple to synthesize and has low cost. In a PB@ZIF-8 / PDA solution, the large specific surface area and high porosity of the ZIF-8 material are utilized to introduce Prussian blue and dopamine hydrochloride respectively, so that ZIF-8 embeds Prussian blue, and dopamine hydrochloride further forms polydopamine (PDA) on the surface of PB@ZIF-8 to accumulate more secondary antibody immunoglobulin G (IgG) to enhance the electrochemical signal. By further functionalizing the electrochemical probe with DON antibody, the competitive immune strategy is used to achieve rapid, sensitive and qualitative detection of DON. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0049] Figure 1 A flow chart of an embodiment of a method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin provided by the present invention;

[0050] Figure 2 A schematic diagram of the principle of the composite electrochemical probe for detecting the concentration of vomitoxin provided by the present invention;

[0051] Figure 3 This is the standard concentration-current curve in the method for detecting the concentration of vomitoxin provided by the present invention.

[0052] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0053] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0054] Traditional detection methods for DON mainly include thin layer chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry, immunoaffinity column purification-high performance liquid chromatography, etc. These methods have defects such as complex sample pre-processing, expensive equipment, complex detection procedures, and the need for professional testing personnel. They are not suitable for the detection of large quantities of samples, and it is difficult to meet the needs of rapid detection results in the grassroots supervision process, and it is not convenient to promote and apply them.

[0055] In view of this, the present invention provides a method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin. The composite electrochemical probe for detecting the concentration of vomitoxin prepared by the preparation method can detect the concentration of vomitoxin quickly, sensitively and stably. Figure 1 The schematic flow chart of one embodiment of a method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin is shown. The method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin comprises the following steps:

[0056] Step S10, zinc nitrate, Prussian blue and 2-methylimidazole are mixed uniformly, centrifuged and filtered to obtain a first precipitate, the first precipitate is mixed with a Tris-HCl buffer solution, and then a dopamine hydrochloride solution is added, the mixture is stirred uniformly, centrifuged and filtered to obtain a second precipitate, and the second precipitate is dispersed in a phosphate buffer to obtain a PB@ZIF-8 / PDA solution;

[0057] Metal organic framework material (ZIF-8) is a type of crystalline porous material with a periodic network structure, and has unique physical and chemical properties, such as rich structure, large specific surface area, high porosity, multiple metal sites, strong catalytic ability, good biocompatibility, good chemical and thermal stability, etc. Therefore, in this embodiment, ZIF-8 material is used to construct an electrochemical sensor, Prussian blue and dopamine hydrochloride are introduced respectively, and Prussian blue is embedded in ZIF-8 material, and dopamine hydrochloride further forms polydopamine on its surface, thereby obtaining PB@ZIF-8 / PDA solution. Since PB@ZIF-8 / PDA solution contains polydopamine, it can accumulate more secondary antibody immunoglobulin G in the subsequent preparation process, thereby enhancing the chemical signal. In this way, the sensitivity of the composite electrochemical probe finally prepared for detecting the concentration of vomitoxin can be improved.

[0058] In addition, in this embodiment, one purpose of using Prussian blue is that it has high catalytic activity and good electrochemical activity, which can improve the detection efficiency. At the same time, Prussian blue itself is simple to synthesize and has low cost.

[0059] Furthermore, the concentrations of zinc nitrate, Prussian blue, 2-methylimidazole, Tris-HCl buffer solution and dopamine hydrochloride solution may also affect the test results. In some embodiments, the concentration of zinc nitrate is 0.1-0.3 mol / L; the concentration of Prussian blue is 1.5-2.5 mmol / L; the concentration of 2-methylimidazole is 0.3-0.5 mol / L; the concentration of Tris-HCl buffer solution is 20-25 mmol / L; and the concentration of dopamine hydrochloride solution is 0.3-0.5 mg / mL.

[0060] Furthermore, in some embodiments, the volume ratio of the zinc nitrate, the Prussian blue and the 2-methylimidazole is 1:(0.8-1.2):(0.8-1.2); that is, 0.8-1.2 mL of Prussian blue and 0.8-1.2 mL of 2-methylimidazole need to be added to every 1 mL of zinc nitrate; as a preferred embodiment of this embodiment, the volume ratio of zinc nitrate, Prussian blue and 2-methylimidazole is 1:1:1.

[0061] Furthermore, in some embodiments, the volume ratio of the Tris-HCl buffer solution to the dopamine hydrochloride solution is 1:(0.8-1.2); that is, for every 1 mL of Tris-HCl buffer solution, 0.8-1.2 mL of dopamine hydrochloride solution needs to be added. As a preferred embodiment of this embodiment, for every 1 mL of Tris-HCl buffer solution, 1 mL of dopamine hydrochloride solution needs to be added.

[0062] When performing step S10, it can be specifically performed by the following steps: mixing zinc nitrate, Prussian blue and 2-methylimidazole in a centrifuge tube, shaking the mixed solution and keeping it at room temperature for 25 to 30 minutes to obtain a suspension, placing the suspension at 2 to 4°C, centrifuging it at a centrifugal speed of 8000 to 11000 rpm for 5 to 15 minutes, centrifuging it 3 to 8 times to obtain a PB@ZIF-8 precipitate, mixing the PB@ZIF-8 precipitate with a Tris-HCl buffer solution, adding a dopamine hydrochloride solution, stirring vigorously for 20 to 30 minutes, and gently shaking it at room temperature for 1.5 to 2 hours to form a suspension, placing the suspension at 3 to 8°C, centrifuging it at a centrifugal speed of 8000 to 11000 rpm for 5 to 15 minutes, centrifuging it 3 to 8 times, and once again obtaining a PB@ZIF-8 precipitate, washing the PB@ZIF-8 precipitate with deionized water and dispersing it in a phosphate buffer to obtain a PB@ZIF-8 / PDA solution.

[0063] It should be noted that the purpose of multiple centrifugal washings is to remove excess solvent and impurities on the surface of the PB@ZIF-8 precipitate and improve the purity of the PB@ZIF-8 precipitate.

[0064] Furthermore, Prussian blue can be prepared by the following steps: 3-5 mmol / L FeCl3 solution and 3-5 mmol / L K4Fe(CN)6 solution are prepared with a hydrochloric acid solution with a pH value of 3, the FeCl3 solution and the K4Fe(CN)6 solution are mixed in a volume ratio of 1:1, stirred evenly at room temperature, centrifuged and washed to obtain a Prussian blue precipitate, and then the Prussian blue precipitate is dispersed in deionized water to obtain a Prussian blue solution.

[0065] The phosphate buffer solution can be prepared by the following steps: 27.386 g of K2HPO4·3H2O, 10.888 g of KH2PO4, and 17.426 g of K2SO4 are simultaneously dissolved in a 1000 mL volumetric flask to make up the volume, and then 0.8 to 1.2 mol / L of sodium hydroxide or 0.8 to 1.2 mol / L of sulfuric acid solution is added to adjust the pH value to 7.0 to 7.4 to obtain a 10 mmol / L phosphate buffer solution.

[0066] Step S20, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to IgG, mixing evenly, to obtain activated IgG.

[0067] When performing step S20, the following steps can be performed: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added to IgG, stirred and mixed, and shaken for 3 to 4 hours to obtain activated IgG.

[0068] The main purpose of step S20 is to activate the carboxyl groups in IgG.

[0069] Furthermore, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.1-0.3 mmol / L; the concentration of N-hydroxysuccinimide is 0.2-0.5 mmol / L; and the concentration of IgG is 0.3-0.5 mg / mL.

[0070] Furthermore, the volume ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and IgG is 1:(0.8-1.2):(20-30); that is, 0.8-1.2 mL of N-hydroxysuccinimide and 20-30 mL of IgG need to be added to every 1 mL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride. As a preferred embodiment of this embodiment, 1 mL of N-hydroxysuccinimide and 25 mL of IgG need to be added to every 1 mL of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride.

[0071] Step S30, adding the activated IgG to the PB@ZIF-8 / PDA solution, incubating, shaking, centrifuging, and filtering to obtain a third precipitate, washing the third precipitate, and obtaining a PB@ZIF-8 / PDA / IgG signal probe.

[0072] It should be noted that, in the present invention, the concentration of vomitoxin is mainly detected by the indirect competition method, that is, the solid phase antigen and the heterogeneous antigen are used to compete for binding to a certain amount of antibody, so the antibody adsorbed on the solid phase is inversely proportional to the content of the antigen to be detected. It is widely used in the fields of fluorescence, photoelectric and electrochemical sensing. Compared with other analytical techniques, electrochemical sensing has the advantages of high sensitivity and rapidity. Its combination with competitive analytical technology provides a broader application prospect for the development of biosensors. Electrochemical immunosensors are a type of biosensor that utilizes the specific binding between antigens and antibodies. It has the advantages of high sensitivity, good selectivity, simple operation, strong specificity, etc., and has good application prospects. In this embodiment, it is mainly through the PB@ZIF-8 / PDA / IgG signal probe and its indirect competitive electrochemical immunosensor, and achieves high-sensitivity detection of vomitoxin.

[0073] When performing step S30, the following steps can be performed: adding the activated IgG to the PB@ZIF-8 / PDA solution, incubating for 30 to 40 minutes, shaking for 8 to 12 hours at 2 to 5°C, obtaining a third precipitate after centrifugation, washing the third precipitate 2 to 3 times with phosphate buffer, adding bovine serum albumin, incubating at 35 to 37°C for 0.8 to 1.2 hours to obtain a fourth precipitate, washing the fourth precipitate 2 to 3 times with phosphate buffer, and dispersing the fourth precipitate in phosphate buffer.

[0074] Furthermore, in some embodiments, the concentration of the bovine serum albumin is 1.0-1.2%; the concentration of the phosphate buffer is 8-10 mmol / L; and the pH value of the phosphate buffer is 7.2-7.5.

[0075] Step S40, grinding and cleaning the electrode, and then drying the electrode with a protective gas to obtain a clean electrode;

[0076] When performing step S40, it can be specifically performed through the following steps: grinding the electrode with sandpaper and alumina powder, washing with deionized water, anhydrous ethanol, and deionized water for 5 to 10 minutes, and then washing with a mixture of hydrogen peroxide and concentrated sulfuric acid for 2 to 3 times to obtain an intermediate product electrode, placing the intermediate product electrode in a sulfuric acid solution, and sequentially subjecting it to constant potential and CV treatment, taking it out and washing it, and then drying it in an inert environment to obtain a clean electrode.

[0077] In some embodiments, the diameter of the aluminum oxide powder is 0.3 μm and / or 0.5 μm; in the mixed solution, the volume ratio of the hydrogen peroxide to the concentrated sulfuric acid is 1:(2.5-3.5); the concentration of the sulfuric acid solution is 0.3-0.5 mol / L; and the protective gas includes nitrogen.

[0078] Specifically, in practice, the electrode is first polished with sandpaper, 0.3 μm and 0.05 μm diameter alumina powder in turn, and then ultrasonically cleaned alternately with deionized water, anhydrous ethanol, and deionized water for 5 to 10 minutes each, a 30% hydrogen peroxide and concentrated sulfuric acid mixture with a volume ratio of 1:3 is prepared, and dripped on the electrode surface for 2 to 5 minutes to remove organic residues, and then the residue of the mixture is washed away with deionized water, and the 30% hydrogen peroxide and concentrated sulfuric acid mixture and deionized water washing steps are repeated twice; the electrode is placed in a 0.5 mol / L H2SO4 solution and treated with constant potential (2 V, 5 s; -0.3 V, 12 s) and CV (-0.1 to 1.5 V, 6 cycles) in turn to remove undissolved impurities; finally, the electrode is washed with a large amount of deionized water, and the surface is dried with nitrogen to prevent water residues, so as to keep the electrode surface clean.

[0079] Step S50, sequentially adding 3,3'-disulfide and DON-bovine serum albumin to the clean electrode to obtain a modified electrode, placing the modified electrode in a BSA solution, and incubating to obtain a DON-BSA / DTSSP / Au electrode.

[0080] When performing step S50, the following steps can be performed: dripping 3,3'-disulfide to the surface of the clean electrode, incubating at 25 to 30°C for 0.5 to 1.5 hours, washing the surface of the clean electrode with a PBST solution, then dripping vomitoxin-bovine serum albumin to the surface of the clean electrode, incubating at 35 to 37°C for 1.5 to 2.5 hours, washing the surface of the clean electrode with a PBST solution to obtain a modified electrode, placing the modified electrode in a BSA solution, incubating for 0.5 to 1.5 hours, and obtaining a DON-BSA / DTSSP / Au electrode.

[0081] Furthermore, in some embodiments, the concentration of the 3,3'-disulfide is 1-3 mmol / L; the concentration of the vomitoxin-bovine serum albumin is 3-5 μg / mL; the concentration of the BSA solution is 1-2%; and the concentration of the DON antibody is 4-5 μg / mL.

[0082] Furthermore, the volume ratio of the 3,3'-disulfide to the DON-BSA is 1:(0.8-1.2); that is, 0.8-1.2 mL of DON-BSA needs to be added for every 1 mL of the 3,3'-disulfide.

[0083] It should be noted that the purpose of adding bovine serum albumin for incubation is to improve the stability of the composite electrochemical probe finally prepared for detecting vomitoxin.

[0084] The PBST solution can be prepared by the following steps: a Tween-20 solution with a volume ratio of 1:2000 is mixed with 10 mmol / L PBS with a pH value of 7.0 to 7.5 at room temperature to obtain a PBST solution.

[0085] Step S60, modifying the DON-BSA / DTSSP / Au electrode with a mixed solution of DON antibodies and DON antigens with different concentration gradients, and then dripping the PB@ZIF-8 / PDA / IgG signal probe to obtain a composite electrochemical probe for detecting DON.

[0086] When performing step S60, it can be specifically performed through the following steps: modifying the DON-BSA / DTSSP / Au electrode with a mixed solution of DON antibodies and DON antigens containing different concentration gradients, placing it at 35 to 37°C, incubating it for 0.5 to 1.5 hours, and then washing it with 10 mmol / L PBST, and then dripping the PB@ZIF-8 / PDA / IgG signal probe onto the DON-BSA / DTSSP / Au electrode, placing it in a constant temperature box at 35 to 37°C, and incubating it for 1.5 to 2.5 hours to obtain a composite electrochemical probe for detecting DON.

[0087] In this example, the concentration of the vomitoxin antibody is 5 μg / mL; the concentrations of the vomitoxin standard are 0.1 pg / mL, 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, 1000 pg / mL, and 5000 pg / mL, respectively.

[0088] In addition, the present invention also provides a method for detecting the concentration of vomitoxin, comprising the following detection steps:

[0089] Step S100, obtaining a working electrode, a reference electrode and an auxiliary electrode, wherein the working electrode comprises a composite electrochemical probe for detecting the concentration of vomitoxin;

[0090] Step S200, placing the working electrode, the reference electrode and the auxiliary electrode in a PBS buffer solution, and obtaining a standard concentration-current curve by differential pulse voltammetry;

[0091] Step S300, modifying the sample to be detected on the working electrode, and then placing the working electrode, the reference electrode and the auxiliary electrode in PBS buffer, obtaining the current curve of the sample to be detected by differential pulse voltammetry, and comparing the current curve of the sample to be detected with the standard concentration-current curve to obtain the vomitin concentration.

[0092] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0093] Example 1

[0094] (1) Preparation of a composite electrochemical probe for detecting vomitoxin concentration

[0095] 600 μL of zinc nitrate (concentration of 0.1 mol / L), 600 μL of Prussian blue (concentration of 1.5 mol / L) and 600 μL of 2-methylimidazole (concentration of 0.3 mol / L) were mixed in a centrifuge tube. The mixture was shaken and kept at room temperature for 30 min to obtain a suspension. The suspension was placed at 4°C and centrifuged at a speed of 11000 rpm for 15 min. The PB@ZIF-8 precipitate was obtained by centrifugation 8 times. 500 μL of the PB@ZIF-8 precipitate was mixed with 1 mL of Tris-HCl buffer solution (concentration of 25 m mol / L) was mixed evenly, and then 500 μL of dopamine hydrochloride solution (concentration of 0.1 mol / L) was added, and after vigorous stirring for 20 min, it was gently shaken at room temperature for 2 h to form a suspension. The suspension was then placed at 8 ° C and centrifuged at a centrifugal speed of 11000 rpm for 15 min. The centrifugation was performed 8 times to obtain a PB@ZIF-8 precipitate again. The PB@ZIF-8 precipitate was washed with deionized water and dispersed in 1 mL of phosphate buffer (concentration of 10 mmol / L, pH value of 7.4) to obtain a PB@ZIF-8 / PDA solution;

[0096] Add 1 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (concentration: 0.2 mmol / L) and 1 μL of N-hydroxysuccinimide (concentration: 0.4 mmol / L) to 25 μL of IgG (concentration: 0.5 mg / mL), stir to mix, and oscillate for 3 to 4 hours to obtain activated IgG.

[0097] The activated IgG was added to the PB@ZIF-8 / PDA solution, incubated for 30 minutes, and then shaken for 10 hours at 4°C, and a third precipitate was obtained after centrifugation. The third precipitate was washed three times with a phosphate buffer (with a concentration of 10 mmol / L and a pH value of 7.4), and then 1.0% bovine serum albumin was added, and the solution was incubated at 37°C for 1 hour to obtain a fourth precipitate. The fourth precipitate was washed three times with a phosphate buffer (with a concentration of 10 mmol / L and a pH value of 7.4), and then the fourth precipitate was dispersed in a phosphate buffer (with a concentration of 10 mmol / L and a pH value of 7.4);

[0098] First, the electrode was polished with sandpaper, 0.3μm and 0.05μm diameter aluminum oxide powder in turn, and then ultrasonically cleaned alternately with deionized water, anhydrous ethanol, and deionized water for 5 minutes each, and a 30% hydrogen peroxide and concentrated sulfuric acid mixture with a volume ratio of 1:3 was prepared and dripped on the electrode surface for 2 minutes to remove organic residues, and then the residue of the mixture was washed off with deionized water, and the 30% hydrogen peroxide and concentrated sulfuric acid mixture and deionized water washing steps were repeated twice; the electrode was placed in a 0.5mol / L H2SO4 solution and treated with constant potential (2V, 5s; -0.3V, 12s) and CV (-0.1~1.5V, 6 cycles) to remove undissolved impurities; finally, the electrode was washed with a large amount of deionized water, and the surface was dried with nitrogen to prevent water residues, so as to keep the electrode surface clean;

[0099] 5 μL of 3,3'-disulfide (concentration of 2 mmol / L) was dropped onto the surface of the clean electrode, incubated at 25°C for 1 h, washed with PBST solution (concentration of 10 mmol / L), and then 5 μL of vomitoxin-bovine serum albumin (5 μg / mL) was dropped onto the surface of the clean electrode, incubated at 37°C for 2 h, washed with PBST solution (concentration of 10 mmol / L) to obtain a modified electrode, and the modified electrode was placed in a BSA solution and incubated for 0.1 h to obtain a DON-BSA / DTSSP / Au electrode;

[0100] The DON-BSA / DTSSP / Au electrode was modified with a mixture of vomitoxin antibodies and vomitoxin antigens with different concentration gradients, incubated at 37°C for 0.1 h, and then washed with 10 mmol / L PBST. Subsequently, the PB@ZIF-8 / PDA / IgG signal probe was added to the DON-BSA / DTSSP / Au electrode, and the electrode was placed in a constant temperature box at 37°C and incubated for 2 h to obtain a composite electrochemical probe for detecting vomitoxin.

[0101] Obtaining a working electrode, a reference electrode and an auxiliary electrode, wherein the working electrode comprises a composite electrochemical probe for detecting the concentration of vomitoxin;

[0102] (2) placing the working electrode, the reference electrode and the auxiliary electrode in a PBS buffer solution, and obtaining a standard concentration-current curve by differential pulse voltammetry;

[0103] (3) The working electrode is modified with commercially available spiked rice flour, and the working electrode, the reference electrode and the auxiliary electrode are placed in a PBS buffer solution. The current curve of the commercially available spiked rice flour is obtained by differential pulse voltammetry, and the current curve of the sample to be tested is compared with the standard concentration-current curve to obtain the vomitorie concentration.

[0104] The test results are shown in Table 1.

[0105] Table 1 Test results of commercially available spiked rice noodles

[0106]

[0107] Among them, mean a The mean value of three measurements, SD b is the standard deviation.

[0108] Example 2

[0109] (1) The same preparation method as step 1;

[0110] (2) placing the working electrode, the reference electrode and the auxiliary electrode in a PBS buffer solution, and obtaining a standard concentration-current curve by differential pulse voltammetry;

[0111] (3) The working electrode is modified with commercially available spiked flour, and the working electrode, the reference electrode and the auxiliary electrode are placed in a PBS buffer solution. The current curve of the sample to be tested is obtained by differential pulse voltammetry, and the current curve of the sample to be tested is compared with the standard concentration-current curve to obtain the vomitorie concentration.

[0112] The test results are shown in Table 2.

[0113] Table 2 Test results of commercially available spiked flour

[0114]

[0115] Among them, mean a The mean value of three measurements, SD b is the standard deviation.

[0116] Example 3

[0117] (1) The same preparation method as step 1;

[0118] (2) placing the working electrode, the reference electrode and the auxiliary electrode in a PBS buffer solution, and obtaining a standard concentration-current curve by differential pulse voltammetry;

[0119] (3) Modifying the working electrode with commercially available corn starch, placing the working electrode, the reference electrode and the auxiliary electrode in a PBS buffer solution, obtaining the current curve of the commercially available corn starch by differential pulse voltammetry, and comparing the current curve of the sample to be tested with the standard concentration-current curve to obtain the vomitorie concentration.

[0120] The test results are shown in Table 3.

[0121] Table 3 Test results of commercially available corn starch

[0122]

[0123] Among them, mean a The mean value of three measurements, SD b is the standard deviation.

[0124] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin, characterized in that: The method comprises the following preparation steps: Mix zinc nitrate, Prussian blue and 2-methylimidazole evenly, centrifuge and filter to obtain a first precipitate, mix the first precipitate with a Tris-HCl buffer solution, add a dopamine hydrochloride solution, stir evenly, centrifuge and filter to obtain a second precipitate, disperse the second precipitate in a phosphate buffer to obtain a PB@ZIF-8 / PDA solution; Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to IgG and mixing well to obtain activated IgG; Adding the activated IgG to the PB@ZIF-8 / PDA solution, incubating, shaking, centrifuging, and filtering to obtain a third precipitate, washing the third precipitate to obtain a PB@ZIF-8 / PDA / IgG signal probe; Grind and clean the electrode, then dry it with protective gas to obtain a clean electrode; Sequentially dropping 3,3'-disulfide and DON-bovine serum albumin onto the clean electrode to obtain a modified electrode, placing the modified electrode in a BSA solution, and incubating to obtain a DON-BSA / DTSSP / Au electrode; The DON-BSA / DTSSP / Au electrode is modified with a mixed solution of DON antibodies and DON antigens containing different concentration gradients, and then the PB@ZIF-8 / PDA / IgG signal probe is added dropwise to obtain a composite electrochemical probe for detecting DON; The steps of adding the activated IgG to the PB@ZIF-8 / PDA solution, incubating, shaking, centrifuging, and filtering to obtain a third precipitate, washing the third precipitate, and obtaining the PB@ZIF-8 / PDA / IgG signal probe include: The activated IgG is added to the PB@ZIF-8 / PDA solution, incubated for 30 to 40 minutes, and then shaken for 8 to 12 hours at 2 to 5° C., and a third precipitate is obtained after centrifugation, and the third precipitate is washed 2 to 3 times with a phosphate buffer, and then bovine serum albumin is added, and the fourth precipitate is incubated at 35 to 37° C. for 0.8 to 1.2 hours to obtain a fourth precipitate, and the fourth precipitate is washed 2 to 3 times with a phosphate buffer, and then the fourth precipitate is dispersed in a phosphate buffer; The steps of sequentially dropping 3,3'-disulfide and DON-bovine serum albumin onto the clean electrode to obtain a modified electrode, placing the modified electrode in a BSA solution, and incubating to obtain a DON-BSA / DTSSP / Au electrode include: 3,3'-disulfide was dropped onto the surface of the clean electrode, and the mixture was incubated at 25-30°C for 0.5-1.5h. The surface of the clean electrode was washed with a PBST solution. Then, vomitoxin-bovine serum albumin was dropped onto the surface of the clean electrode, and the mixture was incubated at 35-37°C for 1.5-2.5h. The surface of the clean electrode was washed with a PBST solution to obtain a modified electrode. The modified electrode was placed in a BSA solution and incubated for 0.5-1.5h to obtain a DON-BSA / DTSSP / Au electrode.

2. The method for preparing the composite electrochemical probe for detecting the concentration of vomitoxin according to claim 1, characterized in that: In the step of uniformly mixing zinc nitrate, Prussian blue and 2-methylimidazole, centrifuging and filtering to obtain a first precipitate, mixing the first precipitate with a Tris-HCl buffer solution, adding a dopamine hydrochloride solution, stirring uniformly, centrifuging and filtering to obtain a second precipitate, and dispersing the second precipitate in a phosphate buffer to obtain a PB@ZIF-8 / PDA solution, The volume ratio of the zinc nitrate, the Prussian blue and the 2-methylimidazole is 1:(0.8-1.2):(0.8-1.2); and / or, The volume ratio of the Tris-HCl buffer solution to the dopamine hydrochloride solution is 1:(0.8-1.2); and / or, The concentration of the zinc nitrate is 0.1-0.3 mol / L; and / or, The concentration of the Prussian blue is 1.5 to 2.5 mmol / L; and / or, The concentration of the 2-methylimidazole is 0.3-0.5 mol / L; and / or, The concentration of the Tris-HCl buffer solution is 20-25 mmol / L; and / or, The concentration of the dopamine hydrochloride solution is 0.3-0.5 mg / mL.

3. The method for preparing the composite electrochemical probe for detecting the concentration of vomitoxin according to claim 1, characterized in that: In the step of adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to IgG and mixing them evenly to obtain activated IgG, The volume ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the N-hydroxysuccinimide and the IgG is 1:(0.8-1.2):(20-30); and / or, The concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.1 to 0.3 mmol / L; and / or, The concentration of the N-hydroxysuccinimide is 0.2-0.5 mmol / L; and / or, The concentration of the IgG is 0.3-0.5 mg / mL.

4. The method for preparing the composite electrochemical probe for detecting the concentration of vomitoxin according to claim 1, characterized in that: The concentration of the bovine serum albumin is 1.0-1.2%; and / or, The concentration of the phosphate buffer is 8 to 10 mmol / L; and / or, The pH value of the phosphate buffer is 7.2-7.

5.

5. The method for preparing the composite electrochemical probe for detecting the concentration of vomitoxin according to claim 1, characterized in that: The steps of cleaning the electrode and then drying the electrode with protective gas to obtain a clean electrode include: The electrode is polished in alumina powder, washed with deionized water, anhydrous ethanol, and deionized water for 5 to 10 minutes, and then washed with a mixture of hydrogen peroxide and concentrated sulfuric acid for 2 to 3 times to obtain an intermediate product electrode. The intermediate product electrode is placed in a sulfuric acid solution, subjected to constant potential and CV treatments in turn, taken out, washed, and dried in an inert environment to obtain a clean electrode.

6. The method for preparing the composite electrochemical probe for detecting the concentration of vomitoxin according to claim 5, characterized in that: The diameter of the aluminum oxide powder is 0.3 μm and / or 0.5 μm; and / or, In the mixed solution, the volume ratio of the hydrogen peroxide to the concentrated sulfuric acid is 1:(2.5-3.5); and / or, The concentration of the sulfuric acid solution is 0.3-0.5 mol / L; and / or, The protective gas includes nitrogen.

7. The method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin according to claim 1, characterized in that: The volume ratio of the 3,3'-disulfide to the DON-BSA is 1:(0.8-1.2); and / or, The concentration of the 3,3'-disulfide is 1 to 3 mmol / L; and / or, The concentration of the DON-BSA is 3-5 μg / mL; and / or, The concentration of the BSA solution is 1-2%; and / or, The concentration of the vomitoxin antibody is 4-5 μg / mL.

8. A method for detecting the concentration of vomitoxin using a composite electrochemical probe prepared by the method for preparing a composite electrochemical probe for detecting the concentration of vomitoxin according to any one of claims 1 to 7, characterized in that: The following detection steps are included: Obtaining a working electrode, a reference electrode and an auxiliary electrode, wherein the working electrode comprises a composite electrochemical probe for detecting the concentration of vomitoxin; The working electrode, the reference electrode and the auxiliary electrode are placed in a PBS buffer solution, and a standard concentration-current curve is obtained by differential pulse voltammetry; The sample to be detected is modified on the working electrode, and then the working electrode, the reference electrode and the auxiliary electrode are placed in PBS buffer, and the current curve of the sample to be detected is obtained by differential pulse voltammetry. The current curve of the sample to be detected is compared with the standard concentration-current curve to obtain the vomitin concentration.

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