Preparation method and application of electrochemical immunosensor for detecting aflatoxin B1

Abstract

The present invention discloses a preparation method and application of an electrochemical immunosensor for detecting aflatoxin B1. In the preparation method provided by the present invention, curcumin is wrapped in ZIF-8 to form CCM@ZIF-8, and then PDA and anti-mouse IgG are modified on the surface of CCM@ZIF8 to obtain an electrochemical nanoprobe. By adding curcumin, the electrochemical nanoprobe is specifically bound to the AFB1 antibody on the surface of the modified electrode to generate an electrochemical signal, thereby realizing the quantitative detection of AFB1, and by embedding it in ZIF-8, its water solubility and stability are improved, so that the stability of the electrochemical nanoprobe is good; by modifying PDA and anti-mouse IgG on the surface of CCM@ZIF8 as signal amplification probes, the sensitivity of the prepared electrochemical immunosensor is high; in addition, the electrochemical immunosensor is simple, fast and has good specificity when used for the determination of AFB1 in grains such as rice noodles.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural product quality and safety detection, and in particular to a preparation method and application of an electrochemical immunosensor for detecting aflatoxin B1. Background Art

[0002] Aflatoxins (AFs) are a group of structurally similar secondary metabolites mainly produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus apis. They are highly toxic and carcinogenic to humans and animals. They are recognized by the World Health Organization as IA carcinogens. They are widely present in moldy agricultural products, food and feed, and seriously endanger human health. Aflatoxins are stable to light, heat and acid, resistant to high temperatures, and have little damage to them due to conventional heating treatments. They are easily enriched in water and soil environments. At present, more than 20 types of aflatoxins have been discovered, and the most common ones are B1, B2, G1, G2, M1 and M2. Among them, aflatoxin B1 (AFB1) is the most toxic and is commonly found in grains such as peanuts, corn, soybeans, wheat and their products. It can cause contamination in the growth, harvesting, storage, transportation, processing, and circulation of grains, posing a huge threat to food safety and human health. In order to monitor aflatoxin contamination in food, ensure food safety and protect the health of consumers, it is necessary to develop accurate and rapid on-site detection technology to monitor AFB1 contamination in food production, processing, circulation and other links.

[0003] The traditional detection method of AFs is mainly chromatography, including thin layer chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry (LC-MS). Chromatography is the gold standard method for detecting AFs in the national standard, with the advantages of accurate detection results, high sensitivity and good repeatability, but it has defects such as complex sample pre-processing, expensive equipment, complex detection procedures, and the need for professional detection personnel. It is 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.

[0004] In order to effectively reduce AFs pollution and ensure food safety, some rapid, accurate and simple AFs detection methods have been developed, such as immunoassay, near-infrared spectroscopy, imaging method and electrochemical biosensor method. Among them, the electrochemical biosensor method refers to the use of biological materials (such as antigens, antibodies, enzymes, DNA, RNA, microorganisms and cells, etc.) as sensitive elements to specifically identify the detection substance, and convert the biochemical signal into electrochemical signals such as current, potential or resistance through a highly sensitive signal converter. Compared with other methods, the electrochemical biosensor method has the advantages of simple operation, high sensitivity and selectivity, wide dynamic range, low detection limit and low cost. However, the electrochemical nanoprobes (i.e., signal markers) of the current electrochemical biosensor are unstable, and the detection results are easily interfered by external factors, resulting in weak signals and low detection sensitivity. Summary of the invention

[0005] The main purpose of the present invention is to propose a preparation method and application of an electrochemical immunosensor for detecting aflatoxin B1, aiming to provide an electrochemical immunosensor with good stability and high sensitivity.

[0006] To achieve the above object, the present invention provides a method for preparing an electrochemical immunosensor for detecting aflatoxin B1, wherein the electrochemical immunosensor comprises a modified electrode, an electrochemical nanoprobe and an AFB1 antibody.

[0007] The preparation method of the electrochemical nanoprobe comprises the following steps:

[0008] S10, Zn 2+ The solution, the curcumin solution, and the 2-methylimidazole solution are mixed to obtain a mixed solution, the mixed solution is shaken, centrifuged to collect a precipitate, and then washed to obtain CCM@ZIF-8;

[0009] S20, dispersing the CCM@ZIF-8 in a solvent, then adding Tris-hydrochloric acid buffer and dopamine solution and mixing, then centrifuging to collect the precipitate, washing, and obtaining an intermediate;

[0010] S30, dispersing the intermediate in a solvent, then adding activated anti-mouse immunoglobulin G, incubating at 35-38° C. for 25-35 min, then shaking at 3-5° C. for 10-14 h, finally centrifuging and collecting the precipitate, washing, and obtaining an electrochemical nanoprobe;

[0011] The preparation method of the modified electrode comprises the following steps:

[0012] A1. Clean the gold electrode, then modify it with DTSSP, then drop the AFB1-BSA solution on the surface of the gold electrode modified with DTSSP, then incubate and clean it, and finally drop the BSA solution for sealing to obtain a modified electrode.

[0013] Optionally, in step S10:

[0014] In the mixed solution, 0.2-0.4 mol of Zn was added for every 0.072 μg of curcumin. 2+ and 0.1 to 0.2 mol of 2-methylimidazole.

[0015] Optionally, step S10 includes:

[0016] The mixed solution was shaken at 35-38° C. for 25-35 min, and then centrifuged at 3-5° C. and 10,000-12,000 r / min for 10-20 min, the precipitate was collected, and the precipitate was washed with methanol for multiple times to obtain CCM@ZIF-8.

[0017] Optionally, the Zn 2+ Zn in solution 2+ The molar ratio of the dopamine in the dopamine solution is 0.2-0.4:0.1; and / or,

[0018] In step S20, in the centrifugation step, the centrifugal speed is 10000-12000 r / min, the centrifugal time is 10-20 min, and the centrifugal temperature is 3-5°C.

[0019] Optionally, for every 0.2-0.4 mol of Zn 2+ Correspondingly, 0.02 mg of anti-mouse immunoglobulin G was added.

[0020] Optionally, step A1 includes:

[0021] The gold electrode was cleaned and then modified with DTSSP. The AFB1-BSA solution was then dropped on the surface of the gold electrode modified with DTSSP. The electrode was then incubated at 35-38°C for 2 h, washed with PBST for several times, and finally BSA solution was added thereto for sealing to obtain a modified electrode.

[0022] Optionally, the diameter of the gold electrode is 2 mm;

[0023] The concentration of the AFB1-BSA solution is 4-6 μg / mL, and the amount of each gold electrode added is 2-4 μL; and / or,

[0024] The concentration of the AFB1 antibody was 5.0 μg / mL.

[0025] Furthermore, the present invention also provides a method for detecting aflatoxin B1, the detection method comprising the following steps: providing an electrochemical immunosensor, the electrochemical immunosensor being prepared by the preparation method of the electrochemical immunosensor for detecting aflatoxin B1 as described above;

[0026] After mixing the AFB1 antibody and the substance to be tested, the mixture is added dropwise to the surface of the modified electrode, incubated at 35-38°C for 50-70 minutes, and then washed with PBST for several times to obtain a first electrode;

[0027] Dispersing the electrochemical nanoprobe in methanol to obtain a probe solution, dropping the probe solution onto the first electrode, incubating at 35-38° C. for 90-120 min, and then washing with PBST to obtain an electrode to be tested;

[0028] The electrode to be tested is used as the working electrode, the saturated mercury electrode is used as the reference electrode, and the platinum wire electrode is used as the counter electrode, and the corresponding current value is detected;

[0029] The AFB1 content in the substance to be tested is calculated according to the current value.

[0030] Optionally, 2 to 4 μL of the AFB1 antibody is added dropwise to each modified electrode; and / or,

[0031] 3 to 4 μL of the probe solution is dripped onto each of the first electrodes.

[0032] Optionally, the substance to be tested includes food and its products.

[0033] The electrochemical immunosensor provided by the present invention is based on the indirect competitive binding between the free AFB1 in the substance to be tested and AFB1-BSA for a fixed number of free AFB1 antibodies, and the current value is detected to detect the AFB1 content in the substance to be tested. The present invention adds curcumin to generate an electrochemical signal after the electrochemical nanoprobe specifically binds to the AFB1 antibody on the surface of the modified electrode, thereby realizing the quantitative detection of AFB1, and by embedding it in ZIF-8 (zeolite imidazole framework 8), its water solubility and stability are improved, so that the stability of the electrochemical nanoprobe is good; by modifying PDA and anti-mouse IgG on CCM@ZIF8, the sensitivity of the electrochemical immunosensor finally prepared is high; in addition, when the electrochemical immunosensor is used for the determination of AFB1 in grains such as rice noodles, it is simple, fast and has good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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 related drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 A schematic flow chart of a method for preparing an electrochemical probe in an electrochemical immunosensor provided by the present invention;

[0036] Figure 2 A schematic diagram of the principle of the electrochemical immunosensor provided by the present invention for detecting aflatoxin B1;

[0037] Figure 3 The SEM images of ZIF-8, CCM@ZIF-8 and CCM@ZIF-8 / PDA prepared in Example 1 of the present invention are shown;

[0038] Figure 4 XRD patterns of simulated ZIF-8, and ZIF-8, CCM@ZIF-8 and CCM@ZIF-8 / PDA in Example 1 of the present invention;

[0039] Figure 5 Thermogravimetric analysis diagrams of CCM, ZIF-8, CCM@ZIF-8, and CCM@ZIF-8 / PDA in Example 1 of the present invention;

[0040] Figure 6 It is the infrared spectra of CCM, ZIF-8, CCM@ZIF-8 and CCM@ZIF-8 / PDA in Example 1 of the present invention;

[0041] Figure 7 The Raman spectra of CCM, ZIF-8, CCM@ZIF-8 and CCM@ZIF-8 / PDA in Example 1 of the present invention are shown;

[0042] Figure 8 The different electrode materials in Example 1 of the present invention are 10mM [Fe (CN) 6] 3- / 4- CV plots of the sensor constructed in solution;

[0043] Fig. 9 The different electrode materials in Example 1 of the present invention were mixed in 5 mM [Fe(CN)6] 3- / 4- EIS graph of the sensor constructed in solution;

[0044] Fig.10CV graphs of the CCM@ZIF-8 / PDA / IgG / Au immunosensor obtained by combining the electrochemical nanoprobe and the gold electrode in Example 1 of the present invention in a 10 mM pH=7.0 PBS solution at different scanning rates with a cumulative time of 20 s;

[0045] Fig.11 This is a graph showing the stability test results of the CCM@ZIF-8 / PDA / IgG electrochemical nanoprobe in Example 1 of the present invention;

[0046] Fig.12 This is a differential pulse voltammogram of the detection of different concentrations of AFB1 in Application Example 1 of the present invention;

[0047] Fig.13 This is a linear relationship diagram between the current and different concentrations of AFB1 drawn in Application Example 1 of the present invention;

[0048] Fig.14 This is a diagram showing the specific analysis results of the electrochemical immunosensor in Application Example 2 of the present invention in different toxins.

[0049] 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

[0050] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0051] In addition, the meaning of "and / or" appearing in the full text includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or a solution that satisfies both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0052] At present, the electrochemical nanoprobes (i.e., signal markers) of electrochemical biosensors are unstable, and the detection results are easily interfered by external factors, resulting in weak signals and low detection sensitivity. According to the different biological materials used in the sensitive elements, electrochemical biosensors are divided into enzyme electrode sensors, microbial sensors, electrochemical immunosensors, etc. Among them, electrochemical immunosensors are designed based on the principle that antibodies have unique recognition and binding functions for corresponding antigens.

[0053] The present invention provides a method for preparing an electrochemical immunosensor for detecting aflatoxin B1. In this embodiment, the electrochemical immunosensor includes a modified electrode, an electrochemical nanoprobe and an AFB1 antibody. Further, the concentration of the AFB1 antibody is 5.0 μg / mL. For ease of description, the AFB1 antibody is abbreviated as Ab-AFB1 below.

[0054] Please refer to Figure 1 In this embodiment, the preparation method of the electrochemical nanoprobe includes the following steps:

[0055] Step S10: Zn 2+ The solution, curcumin solution and 2-methylimidazole solution are mixed to obtain a mixed solution, the mixed solution is shaken, centrifuged to collect the precipitate, and then washed to obtain CCM@ZIF-8.

[0056] Curcumin (CCM), also known as 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, is a natural product extracted from the rhizome of turmeric with low cost. It is also the active ingredient of turmeric and has multiple pharmacological activities, such as anti-cancer, antioxidant, anti-inflammatory, anti-aging, anti-microbial, anti-proliferative, neuroprotective, and cardioprotective. In addition to its well-known biological activities, CCM has also attracted much attention due to its unique chemical structure. CCM has phenolic hydroxyl groups and can undergo redox reactions under electrochemical conditions to gain and lose electrons, but its low water solubility and chemical instability limit its application.

[0057] Metal-organic frameworks (MOFs) are porous nanomaterials self-assembled by metal ions or metal clusters and organic ligands through coordination bonds. Compared with traditional porous materials such as carbon and zeolites, MOFs have the advantages of high porosity, adjustable pore structure, and adjustable metal sites. Zeolite imidazolate framework materials (ZIFs) are an emerging class of MOFs materials that have received widespread attention. Among them, zeolite imidazolate framework 8 (ZIF-8) nanoparticles have outstanding advantages such as high loading capacity, simple synthesis strategy, and good chemical stability.

[0058] Zn 2+The solution and the 2-methylimidazole solution are shaken fully at room temperature to synthesize ZIF-8. In this step, CCM is encapsulated in ZIF-8 to form CCM@ZIF-8 by adopting a biomimetic mineralization method, thereby improving the water solubility and stability of curcumin and making the prepared electrochemical nanoprobe stable.

[0059] Among them, Zn 2+ The solution can be prepared from a water-soluble zinc salt, specifically a zinc chloride solution, a zinc sulfate solution, a zinc acetate solution, and the like.

[0060] Furthermore, the amount of CCM added will affect the detection sensitivity and detection limit of the prepared electrochemical nanoprobe for AFB1. In this embodiment, in the mixed solution, 0.2-0.4 mol of Zn 2+ and 0.1-0.2 mol of 2-methylimidazole. That is, in the mixed solution, Zn 2+ The ratio of the addition amount of curcumin and 2-methylimidazole (2-MI) is 0.2-0.4 mol: 0.072 μg: 0.1-0.2 mol, preferably 0.3 mol: 0.72 μg: 0.18 mol. Under the above ratio, the embedding rate of CCM in ZIF-8 is the highest, which is more than 98%.

[0061] Specifically, the mixed solution is shaken at 35-38° C. for 25-35 min, and then centrifuged at 3-5° C. and 10,000-12,000 r / min for 10-20 min, the precipitate is collected, and the precipitate is washed with methanol for multiple times to obtain CCM@ZIF-8.

[0062] In a preferred embodiment, step S10 includes: adding 300 μL of 1 mol / L Zn 2+ The solution, 120 μL of CCM with a concentration of 6 μg / mL, and 300 μL of 2-MI with a concentration of 0.6 mol / L were mixed in a centrifuge tube to obtain a mixed solution, the mixed solution was oscillated at 37°C for 25 to 35 min, and then centrifuged at 4°C and 11000 r / min for 15 min, the precipitate was collected, and the precipitate was washed three times with methanol to obtain CCM@ZIF-8.

[0063] It was found that as the CCM concentration increased from 2.5μg / mL to 6μg / mL (i.e., when the amount of CCM added was small), the encapsulation efficiency of CCM increased from 46.7% to 98.6%. When the CCM concentration continued to increase (i.e., when the amount of CCM added was high), the encapsulation rate gradually decreased. This is because the diketone structure contained in CCM allows it to form a complex with the metal. When the amount of CCM added was too much, it could compete with 2-MI for Zn 2+Therefore, the optimal CCM concentration is 6 μg / mL.

[0064] Step S20, dispersing the CCM@ZIF-8 in a solvent, then adding Tris-hydrochloric acid buffer and dopamine solution and mixing, then centrifuging to collect the precipitate and washing to obtain an intermediate.

[0065] In this step, three-dimensional polydopamine (PDA) is modified on the surface of CCM@ZIF8 to obtain CCM@ZIF-8 / PDA, i.e., the intermediate. Since PDA has a catechol group, it allows metals to be spontaneously deposited in situ on the surface and has good reduction ability and metal affinity, so it can be used as a multifunctional carrier for signal collection and signal amplification. By adding PDA, the sensitivity of the final bioimmunosensor is improved.

[0066] Among them, every 0.2-0.4 mol of Zn 2+ Correspondingly, 0.02 mg of anti-mouse immunoglobulin G was added. 2 + Zn in solution 2+ The ratio of the amount of dopamine in the dopamine solution is 0.2-0.4 mol: 0.1 mol. In another embodiment, in the centrifugation step, the centrifugal speed is 10000-12000 r / min, the centrifugal time is 10-20 min, and the centrifugal temperature is 3-5°C.

[0067] In a specific embodiment, step S20 includes: dispersing the CCM@ZIF-8 in 1.0 mL of methanol to obtain a CCM@ZIF-8 solution, taking 500 μL of the CCM@ZIF-8 solution and mixing it with 500 μL of a 25 mmol / L Tris-HCl buffer, then adding 500 μL of a 0.1 mol / L dopamine solution, stirring vigorously for 5 minutes, and then shaking at 37° C. for 2 hours to obtain a suspension, centrifuging the suspension at 4° C. and 11,000 r / min for 15 minutes, discarding the supernatant, and washing the collected precipitate with methanol to obtain an intermediate.

[0068] Step S30, dispersing the intermediate in a solvent, then adding activated anti-mouse immunoglobulin G, incubating at 35-38°C for 25-35 minutes, then shaking at 3-5°C for 10-14 hours, finally centrifuging and collecting the precipitate, washing, and obtaining an electrochemical nanoprobe.

[0069] The activated anti-mouse immunoglobulin G is an anti-mouse immunoglobulin G (IgG) activated by EDC / NHS. 2+ Zn in solution2+ The ratio of the added amount to anti-mouse IgG is 0.2-0.4 mol:0.02 mg.

[0070] In this embodiment, the intermediate is dispersed in 1 mL of methanol, and 20 μL of 0.5 mg / mL anti-mouse IgG activated by EDC / NHS is added thereto. The mixture is incubated at 37°C for 30 min, and then shaken at 4°C for 12 h. The supernatant is discarded by centrifugation, and the collected precipitate is washed three times with methanol to remove unbound IgG, thereby obtaining CCM@ZIF-8 / PDA / IgG, i.e., an electrochemical nanoprobe.

[0071] The preparation method of the modified electrode comprises the following steps:

[0072] Step A1, clean the gold electrode, then modify it with DTSSP, then drop the AFB1-BSA solution on the surface of the gold electrode modified with DTSSP, then incubate and clean it, and finally drop the BSA solution for sealing to obtain a modified electrode.

[0073] In this embodiment, the gold electrode is a gold electrode with a diameter of 2 mm.

[0074] The present invention does not limit the specific method of cleaning the gold electrode, and it can be cleaned in a conventional manner. In this embodiment, cleaning the gold electrode includes the following steps: after the gold electrode is polished on suede with Al2O3 powder, it is ultrasonically treated with ultrapure water, anhydrous ethanol and ultrapure water for 5 minutes respectively to remove impurities remaining on the surface of the gold electrode; then, cyclic voltammetry (CV) is used in 0.5M H2SO4 solution to scan 6 to 8 cycles: the potential range is -0.2 to +1.5V, and the scanning rate is 0.1V s -1 ; Finally, [Fe(CN)6] 3- / 4- The gold electrode is cleaned again with the solution, and when the potential difference is less than 100 mV, it indicates that the gold electrode is clean. Preferably, the particle sizes of the Al2O3 powder are 0.3 μm and 0.05 μm, respectively.

[0075] The step of modifying the cleaned gold electrode with DTSSP includes: dropping 3 μL of 2.0 mM 3,3'-dithiobis(sulfonic acid succinimidyl propionate) (DTSSP) on the surface of each gold electrode and incubating at room temperature for 1 hour, and then washing with 10 mM pH 7.0 PBS solution (phosphate buffered saline) three times.

[0076] In this embodiment, step A1 includes: cleaning the gold electrode, then modifying it with DTSSP, then dropping the AFB1-BSA solution on the surface of the gold electrode modified with DTSSP, and then incubating it at 37°C for 2 hours, washing it with PBST for many times, and then adding a BSA-PBST solution with a mass concentration of 1.5% and incubating it at 37°C for 1 hour to block the active site, and finally washing it with PBST three times to obtain a modified electrode.

[0077] In another embodiment, the concentration of the AFB1-BSA solution is 4-6 μg / mL, and the amount added to each gold electrode is 2-4 μL.

[0078] It should be noted that the PBST mentioned in the text is a 10 mM pH 7.0 PBS solution containing 0.05% Tween-20. In addition, BSA is bovine serum albumin.

[0079] The electrochemical immunosensor provided by the present invention is based on the indirect competitive binding between the free AFB1 in the substance to be tested and AFB1-BSA for a fixed number of free AFB1 antibodies, and the current value is detected to detect the AFB1 content in the substance to be tested. The present invention adds curcumin, so that the electrochemical nanoprobe can generate an electrochemical signal after specific binding with the AFB1 antibody on the surface of the modified electrode, and realizes the quantitative detection of AFB1, and by embedding it in ZIF-8 (zeolite imidazole framework 8), its water solubility and stability are improved, so that the stability of the electrochemical nanoprobe is good; by modifying PDA and anti-mouse IgG on the surface of CCM@ZIF8, the sensitivity of the finally prepared electrochemical immunosensor is high; in addition, when the electrochemical immunosensor is used for the determination of AFB1 in food and its products, it is simple, fast and has good specificity.

[0080] In addition, the present invention also proposes a method for detecting aflatoxin B1, which comprises the following steps:

[0081] Step B1, providing an electrochemical immunosensor, wherein the electrochemical immunosensor is prepared by the preparation method of the electrochemical immunosensor for detecting AFB1 as described above.

[0082] Step B2: After mixing the AFB1 antibody and the substance to be tested, drip them onto the surface of the modified electrode, incubate them at 35-38° C. for 50-70 min, and then wash them with PBST for several times to obtain a first electrode.

[0083] The incubation time of Ab-AFB1 (AFB1 antibody) is an important parameter in the competitive format, because the fixed amount of AFB1-BSA on the electrode interface and the free AFB1 in the solution will have a competitive immune reaction with Ab-AFB1. Preferably, the incubation time of Ab-AFB1 is 50 minutes. Under the above incubation time, the current value is the largest and time is saved.

[0084] Furthermore, 2 to 4 μL of Ab-AFB1 is added dropwise to each modified electrode, preferably 3 μL.

[0085] The present invention does not limit the specific type of the substance to be tested, which may be soy sauce, edible oil, etc. In this embodiment, the substance to be tested is food and its products, specifically rice noodles, flour, etc. Since food is a complex system containing many other toxic substances that interact with each other, it is very important to ensure that the developed electrochemical immunosensor maintains good selectivity for AFB1 in the presence of food matrix interfering molecules. The electrochemical immunosensor provided by the present invention has good specificity for AFB1 and has obvious competitive advantages.

[0086] Step B3, dispersing the electrochemical nanoprobe in methanol to obtain a probe solution, dropping the probe solution onto the first electrode, incubating at 35-38° C. for 90-120 min, and then washing with PBST to obtain the electrode to be tested.

[0087] Preferably, the probe solution is dripped onto the first electrode and incubated at 37°C for 90 minutes. In addition, too little or too much probe solution will cause the droplets to fail to stably bind to the modified electrode, resulting in a low current value. In this embodiment, 3 to 4 μL of the probe solution is dripped onto each of the first electrodes, preferably 3 μL. Under the above dripping amount, the electrochemical nanoprobe can form a stable sphere on the surface of the Au electrode, which is not easy to fall off.

[0088] Step B4: using the electrode to be tested as the working electrode, the saturated mercury electrode as the reference electrode, and the platinum wire electrode as the counter electrode, to detect and obtain the corresponding current value.

[0089] Step B5, calculating the AFB1 content in the substance to be tested according to the current value.

[0090] Of course, before detecting the substance to be tested, it is necessary to first establish a standard curve of the corresponding relationship between the current value and the AFB1 content, and then calculate the AFB1 content in the substance to be tested based on the standard curve and the current value of the substance to be tested.

[0091] In specific implementation, the detection method includes: (1) using the electrochemical immunosensor as described above; (2) mixing 3 μL of Ab-AFB1 and 3 μL of AFB1 standard solution of different concentrations to obtain a mixed solution, and dripping the mixed solution onto the surface of different modified electrodes, one concentration of AFB1 standard solution corresponds to one modified electrode, incubating at 37° C. for 50 minutes, and then washing with PBST for multiple times to obtain a first electrode; (3) dispersing the electrochemical nanoprobe in methanol to obtain a probe solution, dripping 3 μL of the probe solution onto each first electrode, incubating at 37° C. for 90 minutes, and then washing with PBST to obtain an electrode to be tested; (4) constructing a standard curve: using the above-mentioned electrode to be tested as the working electrode, the saturated mercury electrode as the reference electrode, and the platinum wire electrode as the counter electrode, the electrochemical signal detected by the CHI660E electrochemical workstation; at 10 mM The test was carried out in a PBS solution with a pH of 7; the scanning voltage range was 0-0.4V, the amplitude was 0.005V, and the frequency was 20Hz; each concentration of the AFB1 standard solution corresponded to a current value, and a standard curve was constructed based on the logarithm of the current value and the concentration of AFB1; (5) Detection of AFB1 in the test substance: first obtain the test solution, and then use the same method as the test AFB1 standard solution to obtain the current value of the test electrode corresponding to the test solution; substitute the current value into the standard curve to obtain the concentration of AFB1 in the test solution.

[0092] Please refer to Figure 2 The electrochemical immunosensor adopts indirect competitive immunoassay, which is based on the indirect competitive binding between the free AFB1 in the substance to be tested and AFB1-BSA for a fixed amount of free Ab-AFB1, and detects the current value to detect the AFB1 content in the substance to be tested. The specific detection principle is as follows:

[0093] If a sample contaminated by AFB1 is added to the system, once the concentration of AFB1 in the sample is excessive, there will be no excess free Ab-AFB1 that can bind to AFB1-BSA, and Ab-AFB1 will bind to the free AFB1 in the sample. Since CCM@ZIF-8 / PDA / IgG has no binding sites on the surface of the modified electrode, it will be removed during the washing process, so its electrochemical signal value is low; if a sample not contaminated by AFB1 is added to the system, Ab-AFB1 will bind to AFB1-BSA on the surface of the modified electrode, and the electrochemical nanoprobe can specifically capture Ab-AFB1, which will not be removed during the washing process, thus generating a higher electrochemical signal value.

[0094] 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.

[0095] Example 1 Electrochemical Immunosensor

[0096] The electrochemical immunosensor comprises a modified electrode, an electrochemical nanoprobe and an AFB1 antibody.

[0097] The preparation method of the electrochemical nanoprobe comprises the following steps:

[0098] (1) Add 300 μL of 1 mol / L Zn 2+ The solution, 120 μL of CCM with a concentration of 6 μg / mL, and 300 μL of 2-MI with a concentration of 0.6 mol / L were mixed in a centrifuge tube to obtain a mixed solution (i.e., in the mixed solution, Zn 2+ , curcumin and 2-methylimidazole were added in a ratio of 0.3 mol: 0.072 μg: 0.18 mol), the mixture was shaken at 37° C. for 30 min, and then centrifuged at 4° C. and 11000 r / min for 15 min, the precipitate was collected, and the precipitate was washed with methanol three times to obtain CCM@ZIF-8;

[0099] (2) The above CCM@ZIF-8 was dispersed in 1.0 mL of methanol to obtain a CCM@ZIF-8 solution, 500 μL of the CCM@ZIF-8 solution was mixed with 500 μL of a 25 mmol / L Tris-HCl buffer, and then 500 μL of a 0.1 mol / L dopamine solution was added. After vigorous stirring for 5 min, the suspension was shaken at 37°C for 2 h to obtain a suspension. The suspension was centrifuged at 4°C and 11000 r / min for 15 min, the supernatant was discarded, and the collected precipitate was washed with methanol to obtain a CCM@ZIF-8 / PDA composite material, i.e., an intermediate, i.e., Zn 2+ Zn in solution 2+ The molar ratio of the dopamine in the dopamine solution is 0.3 mol:0.1 mol;

[0100] (3) The intermediate was dispersed in 1 mL of methanol, and 20 μL of 0.5 mg / mL EDC / NHS activated anti-mouse IgG was added thereto (i.e., Zn 2+ Zn in solution 2+ The ratio of added amount to anti-mouse IgG was 0.3 mol: 0.02 mg), incubated at 37°C for 30 min, and then shaken at 4°C for 12 h. Finally, the supernatant was discarded by centrifugation, and the collected precipitate was washed three times with methanol to remove unbound IgG to obtain CCM@ZIF-8 / PDA / IgG, i.e., the electrochemical nanoprobe.

[0101] The preparation method of the modified electrode comprises the following steps:

[0102] (1) A gold electrode with a diameter of 2 mm was polished on suede with Al2O3 powder and then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water for 5 minutes each to remove impurities remaining on the surface of the gold electrode. Cyclic voltammetry (CV) was then performed in 0.5 M H2SO4 solution for 6 to 8 cycles: the potential range was -0.2 to +1.5 V, and the scan rate was 0.1 V s -1 ; Finally, [Fe(CN)6] 3- / 4- The solution is used to clean the gold electrode again. When the potential difference is less than 100mV, it indicates that the gold electrode is clean.

[0103] (2) After each cleaned gold electrode, 3 μL of 2.0 mM DTSSP was added and incubated at room temperature for 1 h, then washed three times with 10 mM pH 7.0 PBS solution, and then 3 μL of 5 μg / mL AFB1-BSA was added to each electrode, and then incubated at 37°C for 2 h, washed three times with PBST, and then 1.5% BSA-PBST solution was added and incubated at 37°C for 1 h to block the active sites, and finally washed three times with PBST to obtain a modified electrode.

[0104] The concentration of the AFB1 antibody was 5.0 μg / mL.

[0105] 1. Morphology characterization

[0106] The morphology of CCM, ZIF-8, CCM@ZIF-8 and CCM@ZIF-8 / PDA provided or prepared by the preparation method of this embodiment was characterized by the following specific tests:

[0107] (1) SEM characterization:

[0108] The morphological characteristics of ZIF-8, CCM@ZIF-8 and CCM@ZIF-8 / PDA were studied using SEM. Figure 3 (A) and Figure 3 (B) SEM images of ZIF-8 and CCM@ZIF-8. Figure 3 It can be seen that the average particle sizes of ZIF-8 and CCM@ZIF-8 are (100±3.8) nm and (100±3.4) nm, respectively. ZIF-8 presents a typical rhombic dodecahedron morphology, and its morphology does not change significantly after being embedded in CCM. Figure 3 As can be seen from (C), the morphology of CCM@ZIF-8 / PDA has no obvious change compared with ZIF-8 and CCM@ZIF-8, and the average particle size is (100±4.2) nm, indicating that the modification of CCM and PDA did not destroy the structure of ZIF-8.

[0109] (2) XRD characterization:

[0110] The XRD patterns of simulated ZIF-8, ZIF-8, CCM@ZIF-8 and CCM@ZIF-8 / PDA nanocomposites are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that for pure ZIF-8, diffraction peaks were observed at 2θ = 7.26°, 14.7°, 16.3°, 17.6°, 21.8°, 24.2°, 26.2°, 29.7°, and 30.6°, which is similar to the simulated pure ZIF-8. After adding CCM and PDA, the physicochemical properties of ZIF-8 remain unchanged, and the results between the two are basically consistent, thus indicating that CCM and PDA do not affect the crystal structure of ZIF-8.

[0111] (3) Thermogravimetric characterization:

[0112] The thermal stability of CCM, ZIF-8, CCM@ZIF-8, and CCM@ZIF-8 / PDA was studied by thermogravimetric analysis. Figure 5 As shown by Figure 5 It can be seen that with the increase of temperature, CCM begins to lose weight and degrade at 200°C. ZIF-8 begins to gradually lose weight at 190°C, corresponding to the removal of guest molecules (mainly H2O) or unreacted substances (such as 2-MI) in the cavity from the surface of the nanocrystal. The relative mass is stable for a long time in the range of 200-500°C, indicating that ZIF-8 has good thermal stability. Both CCM@ZIF-8 and CCM@ZIF-8 / PDA have good thermal stability, and their TGA curves show gradual decomposition from 150-400°C, which is caused by the degradation of proteins in the composite material. CCM@ZIF-8 / PDA gradually decomposes at 500-700°C mainly to remove unreacted PDA. It shows that the stability of CCM is improved by embedding it in ZIF-8.

[0113] (4) Infrared spectroscopy characterization:

[0114] In order to verify the synthesis of CCM@ZIF-8 / PDA composites, infrared spectroscopy was used to analyze CCM, ZIF-8, CCM@ZIF-8 and CCM@ZIF-8 / PDA. Figure 6 As shown. Figure 6 It can be seen that in the CCM infrared spectrum, the most significant carbonyl region (1800-1650cm -1 ) has no band, indicating that CCM exists in the form of keto-enol tautomerism. CCM spectrum at 3509cm -1 There is an obvious peak at 1628cm, which is caused by the stretching vibration of phenol-OH. -1 (C=O and C=C vibration), 1604cm -1(CCM benzene ring stretching vibration), 1508cm -1 (CO and CC vibration), 1429cm -1 (olefin CH bending vibration), 1282cm -1 (aromatic CO stretching vibration) and 1026cm -1 A strong peak appeared at 450-800 cm (COC stretching vibration). -1 The peak at 1350-1500cm is the bending vibration of the imidazole ring. -1 The band is related to the stretching of the entire 2-MI. Among them, 422cm -1 (Zn-N stretching mode), 1146cm -1 (CN stretch mode), 1584cm -1 (C=N stretching mode) and 2929cm -1 and 3136cm -1 The peak of (CH stretching mode) is consistent with that reported previously. However, there is no CCM at 3509 cm in ZIF@CCM. -1 The phenolic hydroxyl stretching vibration peak at 1634 is because most of the CCM in ZIF@CCM is encapsulated in the ZIF-8 framework, and less CCM is adsorbed on the surface. In the CCM@ZIF-8 / PDA graph, it can be seen that other new absorption bands appear at 1634 and 1384. Compared with CCM@ZIF-8, these new absorption bands are obviously the characteristics of PDA. This result further proves that PDA is successfully set on the CCM@ZIF-8 surface.

[0115] (5) Raman characterization:

[0116] In order to further verify the synthesis of CCM@ZIF-8 / PDA composites, Raman spectroscopy was used for characterization. Figure 7 Raman spectra of CCM, ZIF-8, CCM@ZIF-8 and CCM@ZIF-8 / PDA composites. Figure 7 Medium, 1500cm -1 The absorption peak is mainly derived from the vibration of C=C bond, 1550cm -1 ~1580cm -1 Mainly N=N bond vibration, 2800cm -1 ~3000cm -1 The spectral band is mainly due to the vibration of the CH bond. In addition, the 687cm -1 and 1142cm -1 There is a strong absorption band at 1680cm, which comes from imidazole and Zn-N, indicating that ZIF-8 was successfully synthesized. -1 ~1820cm-1 The band at 550 cm-1 corresponds to the amide I band, which is mainly derived from the C=O stretching mode, indicating the presence of CCM in the composite. -1 ~800cm -1 The band at 1610 cm is mainly due to the vibration of C-Cl bond. -1 ~1680cm -1 The vibration of the spectral band is mainly the vibration of C=N bond, indicating that PDA has been successfully modified onto CCM@ZIF-8. The results show that CCM@ZIF-8 and CCM@ZIF-8 / PDA nanocomposites were successfully synthesized.

[0117] 2. Electrochemical behavior of electrodes of different materials

[0118] The electrochemical test was carried out in a CHI660E electrochemical workstation. The electrochemical test used a traditional three-electrode system, with a saturated calomel electrode as the reference electrode, a platinum sheet as the counter electrode, and a gold electrode as the working electrode. The electrochemical behavior of the electrochemical immunosensor constructed in Example 1 was investigated by CV and EIS methods, respectively. The results are shown in Figure 2. Figure 8 and Fig. 9 shown.

[0119] Figure 8 For 10 mM [Fe(CN)6] 3- / 4- CV plots of sensors constructed with different electrode materials in solution. Figure 8 In the figure, a represents Au, b represents DTSSP / Au, c represents AFB1-BSA / DTSSP / Au, d represents BSA / AFB1-BSA / DTSSP / Au, e represents Ab-AFB1 / BSA / AFB1-BSA / DTSSP / Au, and f represents CCM@ZIF-8 / PDA / IgG / Ab-AFB1 / BSA / AFB1-BSA / DTSSP / Au.

[0120] Depend on Figure 6 It can be seen that after adding DTSSP to the surface of the Au electrode, the peak current (curve b) is lower than that of bare Au (curve a), indicating that the added DTSSP can increase [Fe(CN)6] 3- / 4-The electron transfer to the electrode causes the current to decrease; after AFB1-BSA is dropped onto the electrode, the peak current decreases significantly, indicating that AFB1-BSA is successfully fixed on the DTSSP / Au surface (curve c); after adding BSA, the peak current continues to decrease (curve d), indicating that BSA has a good sealing effect on AFB1-BSA / DTSSP / Au; after Ab-AFB1 is modified on the fabricated immunosensor, the peak current continues to decrease with the increase of transfer resistance (curve e), and this result shows that Ab-AFB1 successfully binds to AFB1 on the modified electrode; finally, when the electrochemical nanoprobe is dropped onto the modified electrode, the peak current reaches the minimum value (curve f), indicating that the electrochemical nanoprobe is successfully fixed on the immunosensor.

[0121] Fig. 9 In 5 mM [Fe(CN)6] containing 0.1 M KCl 3- / 4- EIS graphs of sensors constructed with different electrode materials in solution. EIS is an effective detection method, which is usually used to detect the interfacial properties of the surface film of the modified electrode. The radius of EIS reflects the resistance of the modified electrode. The larger the radius, the greater the electrode resistance, which reflects the difficulty of the charge transfer process of the modified layer.

[0122] Depend on Fig. 9 It can be seen that the bare Au (a) electrode R ct The EIS graph is a straight line. ct Much larger than bare Au (a), this is because DTSSP can form Au-S with Au, resulting in R ct For AFB1-BSA / DTSSP / Au(c) and BSA / AFB1-BSA / DTSSP / Au(d), R ct The resistance of the Au electrode was increased as DTSSP, as a stable protein cross-linker, could bind to AFB1-BSA, leading to an increase in the resistance shift rate. As a blocking agent, BSA led to an increase in resistance, which proved that AFB1-BSA was completely bound to the Au electrode surface. After coating Ab-AFB1 on the BSA / AFB1-BSA / DTSSP / Au surface, the R of Ab-AFB1 / BSA / AFB1-BSA / DTSSP / Au(e) was ct The R ct The results show that the Ab-AFB1 is successfully captured by the electrochemical nanoprobe, which increases the electron transfer rate of the composite material. The results show that the electrochemical immunosensor provided by the present invention does work according to the principle described above.

[0123] 3. Kinetic analysis

[0124] Fig.10 The CCM@ZIF-8 / PDA / IgG / Au immunosensor obtained by combining the electrochemical nanoprobe and the gold electrode in Example 1 was tested in a PBS (pH = 7.0) solution at different scan rates (100, 125, 150, 175, 200, 225, 250, 275 and 300 mV s -1 ), CV graph with a cumulative time of 20s. Fig.10 It can be seen that as the scan rate increases from 100 to 300 mV -1 , redox peak current (I pa and I pc ) has a good linear relationship with the scanning rate (v). The linear relationship equations are: pa =0.0034v+0.0791(R 2 =0.9987) and I pc =0.0021v-0.2158(R 2 =0.9860)( Fig.10 ), indicating that the electrochemical behavior of CCM@ZIF-8 / PDA / IgG / Au is an adsorption-controlled process. At the same time, it indicates that the electrochemical nanoprobe provided by the present invention has a good redox peak and can cause changes in current by gaining and losing electrons.

[0125] 4. Stability test

[0126] The long-term stability of the CCM@ZIF-8 / PDA / IgG electrochemical nanoprobe prepared in Example 1 was studied, and the results are as follows: Fig.11 As shown. The electrochemical nanoprobe was stored in a refrigerator at 4°C and measured every 10 days, for a total of 5 times. The stability of the immunosensor was evaluated based on the change in the probe current value. In the first 10 days, the current signal changed from 0.3979μA to 0.3710μA. In the next 3 days, the currents were 0.3551μA, 0.3463μA and 0.3300μA, respectively. After 50d, the current dropped by 0.0679μA. The relative standard deviation (RSDs) was 4.8% (n=3), indicating that the electrochemical nanoprobe provided by the present invention has good long-term stability.

[0127] Example 2

[0128] The electrochemical immunosensor comprises a modified electrode, an electrochemical nanoprobe and an AFB1 antibody.

[0129] The preparation method of the electrochemical nanoprobe comprises the following steps:

[0130] (1) Add 200 μL of 1 mol / L Zn 2+ The solution, 120 μL of CCM with a concentration of 6 μg / mL, and 400 μL of 2-MI with a concentration of 0.25 mol / L were mixed in a centrifuge tube to obtain a mixed solution (i.e., in the mixed solution, Zn 2+ , curcumin and 2-methylimidazole in an amount ratio of 0.2 mol: 0.072 μg: 0.1 mol), the mixture was shaken at 37° C. for 25 to 35 min, and then centrifuged at 3° C. and 12,000 r / min for 10 min, the precipitate was collected, and the precipitate was washed three times with methanol to obtain CCM@ZIF-8;

[0131] (2) The above CCM@ZIF-8 was dispersed in 1.0 mL of methanol to obtain a CCM@ZIF-8 solution. 500 μL of the CCM@ZIF-8 solution was mixed with 500 μL of a 25 mmol / L Tris-HCl buffer solution, and then 500 μL of a 0.1 mol / L dopamine solution was added. After vigorous stirring for 5 min, the mixture was slightly shaken at 37°C for 2 h to obtain a suspension. The suspension was centrifuged at 3°C ​​and 12,000 r / min for 10 min, the supernatant was discarded, and the collected precipitate was washed with methanol to obtain an intermediate, namely: Zn 2+ Zn in solution 2+ The molar ratio of the dopamine in the dopamine solution is 0.2 mol:0.1 mol;

[0132] (3) The intermediate was dispersed in 1 mL of methanol, and 20 μL of 0.5 mg / mL EDC / NHS activated anti-mouse IgG was added thereto (i.e., Zn 2+ Zn in solution 2+ The ratio of added amount to anti-mouse IgG was 0.3 mol: 0.02 mg), incubated at 35°C for 35 min, and then shaken at 3°C ​​for 10 h. Finally, the supernatant was discarded by centrifugation, and the collected precipitate was washed three times with methanol to remove unbound IgG to obtain CCM@ZIF-8 / PDA / IgG, i.e., the electrochemical nanoprobe.

[0133] The preparation method of the modified electrode comprises the following steps:

[0134] (1) A gold electrode with a diameter of 2 mm was polished on suede with Al2O3 powder and then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water for 5 minutes each to remove impurities remaining on the surface of the gold electrode. Cyclic voltammetry (CV) was then performed in 0.5 M H2SO4 solution for 6 to 8 cycles: the potential range was -0.2 to +1.5 V, and the scan rate was 0.1 V s -1 ; Finally, [Fe(CN)6]3- / 4- The solution is used to clean the gold electrode again. When the potential difference is less than 100mV, it indicates that the gold electrode is clean.

[0135] (2) After each cleaned gold electrode, 3 μL of 2.0 mM DTSSP was added and incubated at room temperature for 1 h, then washed three times with 10 mM pH 7.0 PBS solution, and then 2 μL of 4 μg / mL AFB1-BSA was added to each electrode, and then incubated at 37°C for 2 h, washed three times with PBST, and then 1.5% BSA-PBST solution was added and incubated at 37°C for 1 h to block the active sites, and finally washed three times with PBST to obtain a modified electrode.

[0136] The concentration of the AFB1 antibody was 5.0 μg / mL.

[0137] Example 3

[0138] The electrochemical immunosensor comprises a modified electrode, an electrochemical nanoprobe and an AFB1 antibody.

[0139] The preparation method of the electrochemical nanoprobe comprises the following steps:

[0140] (1) Add 400 μL of 1 mol / L Zn 2+ The solution, 120 μL of CCM with a concentration of 6 μg / mL, and 500 μL of 2-MI with a concentration of 0.5 mol / L were mixed in a centrifuge tube to obtain a mixed solution (i.e., in the mixed solution, Zn 2+ , curcumin and 2-methylimidazole in an amount ratio of 0.4 mol: 0.072 μg: 0.2 mol), the mixture was shaken at 37° C. for 25 to 35 min, and then centrifuged at 5° C. and 10,000 r / min for 20 min, the precipitate was collected, and the precipitate was washed three times with methanol to obtain CCM@ZIF-8;

[0141] (2) The above CCM@ZIF-8 was dispersed in 1.0 mL of methanol to obtain a CCM@ZIF-8 solution. 500 μL of the CCM@ZIF-8 solution was mixed with 500 μL of a 25 mmol / L Tris-HCl buffer solution, and then 500 μL of a 0.1 mol / L dopamine solution was added. After vigorous stirring for 5 min, the mixture was slightly shaken at 37° C. for 2 h to obtain a suspension. The suspension was centrifuged at 5° C. and 10,000 r / min for 20 min, the supernatant was discarded, and the collected precipitate was washed with methanol to obtain an intermediate, namely: Zn 2+ Zn in solution 2+ The molar ratio of the dopamine in the dopamine solution is 0.4 mol:0.1 mol;

[0142] (3) The intermediate was dispersed in 1 mL of methanol, and 20 μL of 0.5 mg / mL EDC / NHS activated anti-mouse IgG was added thereto (i.e., Zn 2+ Zn in solution 2+ The added ratio of anti-mouse IgG was 0.3 mol: 0.02 mg), incubated at 38°C for 25 min, and then shaken at 5°C for 14 h. Finally, the supernatant was discarded by centrifugation, and the collected precipitate was washed three times with methanol to remove unbound IgG to obtain CCM@ZIF-8 / PDA / IgG, i.e., the electrochemical nanoprobe.

[0143] The preparation method of the modified electrode comprises the following steps:

[0144] (1) A gold electrode with a diameter of 2 mm was polished on suede with Al2O3 powder and then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water for 5 minutes each to remove impurities remaining on the surface of the gold electrode. Cyclic voltammetry (CV) was then performed in 0.5 M H2SO4 solution for 6 to 8 cycles: the potential range was -0.2 to +1.5 V, and the scan rate was 0.1 V s -1 ; Finally, [Fe(CN)6] 3- / 4- The solution is used to clean the gold electrode again. When the potential difference is less than 100mV, it indicates that the gold electrode is clean.

[0145] (2) After each cleaned gold electrode, 3 μL of 2.0 mM DTSSP was added and incubated at room temperature for 1 h, then washed three times with 10 mM pH 7.0 PBS solution, and then 2 μL of 6 μg / mL AFB1-BSA was added to each electrode, and then incubated at 37°C for 2 h, washed three times with PBST, and then 1.5% BSA-PBST solution was added and incubated at 37°C for 1 h to block the active sites, and finally washed three times with PBST to obtain a modified electrode.

[0146] The concentration of the AFB1 antibody was 5.0 μg / mL.

[0147] Application Example 1 Detection of Aflatoxin B1

[0148] The electrochemical immunosensor described in Example 1 was used for detection, and the specific method was as follows:

[0149] (1) Dissolve different amounts of AFB1 standard in 10 mM pH 7.0 PBS solution to obtain AFB1 standard solutions with concentrations of 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, 1 ng / mL, 5 ng / mL, and 10 ng / mL, respectively, for later use;

[0150] (2) 3 μL of AFB1 standard solutions of different concentrations were mixed with 3 μL of AFB1 antibody with a concentration of 5 μg / mL to obtain multiple groups of mixed solutions, and the multiple groups of mixed solutions were dripped onto the surface of the modified electrode (three modified electrodes were set in each group for parallel experiments), one group of mixed solutions corresponded to one modified electrode (i.e., one concentration of AFB1 standard solution corresponded to one modified electrode), and incubated at 37°C for 50 min, and then washed three times with 10 mM pH 7.4 PBST to obtain the first electrode;

[0151] (3) dispersing the electrochemical nanoprobe in 1 mL of methanol to obtain a probe solution, dropping 3 μL of the probe solution on the surface of each of the first electrodes, and incubating at 37° C. for 90 min to obtain an electrode to be tested;

[0152] (4) Using a conventional three-electrode system, the electrode to be tested was subjected to differential pulse voltammetry in a 10 mM pH 7.0 PBS solution and measured in the voltage range of 0 to 0.4 V. The following results were obtained: Fig.12 The differential pulse voltammogram shown in Fig.13 Shown is a standard curve of current versus aflatoxin AFB1 concentration.

[0153] Depend on Fig.13 It can be seen that in the range of 0.5pg / mL to 10ng / mL, the immunoreaction current value I decreases with the increase of AFB1 concentration, and has a good linear relationship. The linear equation is I = -0.0569logc AFB1 +0.3699, R 2 =0.9954 (e.g. Fig.13 ), and the minimum detection limit is 0.46 pg / mL (S / N=3). The data show that the AFB1 electrochemical immunosensor provided by the present invention can successfully detect low concentrations of AFB1, and has a wide linear range and a low detection limit.

[0154] Application Example 2 Specificity Analysis

[0155] The same method as in Application Example 1 was used to detect AFB1 structurally similar toxins AFB2, AFG1, AFG2 and DON. Under the same conditions, the concentration of AFB1 to be detected was 10 pg / mL, and the concentration of other mycotoxins was 100 pg / mL. The test results are as follows Fig.14 shown.

[0156] Depend on Fig.14 It can be seen that, except for AFB1, the current values ​​of the other four interfering toxins at a concentration of 100 pg / mL are the same as those of the blank sample. -1 ) was 10 times lower, but the current value was significantly reduced. After significance analysis, there was no statistically significant difference in the current of AFB2, AFG1, AFG2, DON and the blank group (P<0.001). Therefore, this result strongly proves that the electrochemical immunosensor prepared by the electrochemical nanoprobe based on CCM@ZIF-8 / PDA / IgG has good specificity for detecting AFB1.

[0157] Application Example 3 Spiked Recovery Test in Rice Flour

[0158] The electrochemical immunosensor described in Example 1 was used for detection, and the specific method was as follows:

[0159] (1) Take 4 g of rice flour sample powder, add 10 mL of methanol-water extract, shake vigorously for 5 min to fully extract the sample powder, centrifuge at 6000 rpm / min for 20 min, and filter three times with an organic syringe filter; take 1 mL of the filtrate and dilute it with 5 mL of phosphate buffer to obtain a reserve solution for standby use, wherein the volume ratio of methanol to water in the methanol-water extract is 8:2. Take the reserve solution and mix it with AFB1 standard solutions with concentrations of 10 pg / mL, 200 pg / mL, and 1 ng / mL in a volume ratio of 1:1, respectively, to obtain 3 sets of sample spike solutions for standby use;

[0160] (2) 3 μL of sample spike solution was mixed with 3 μL of 5 μg / mL antibody working solution to obtain three mixed solutions. The three mixed solutions were added dropwise to the surfaces of three modified electrodes (three modified electrodes were set in each group for parallel experiments). One modified electrode corresponded to one mixed solution and was incubated at 37°C for 50 min. Then, it was washed three times with 10 mM pH 7.4 PBST to obtain the first electrode.

[0161] (3) dispersing the electrochemical nanoprobe in 1 mL of methanol to obtain a probe solution, dropping 3 μL of the probe solution on the surface of each of the first electrodes, and incubating at 37° C. for 90 min to obtain an electrode to be tested;

[0162] (4) Using an electrochemical workstation to detect the current value to be measured;

[0163] (5) Calculate the AFB1 concentration in the spiked sample based on the standard curve in Application Example 1.

[0164] The test results showed that the concentrations of AFB1 in the three rice noodle spiked samples were 4.06±0.09pg / mL, 100.33±2.79pg / mL and 461.59±7.20pg / mL, respectively, and the recovery rates were between 81% and 100%, indicating that the method of the present invention can be used for the detection of aflatoxin AFB1 in rice noodles.

[0165] Application Example 4 Spiked Recovery Test in Flour

[0166] Except that rice flour is replaced by flour, the remaining steps are the same as those in Application Example 3.

[0167] The test results showed that the concentrations of AFB1 in the three flour spiked samples were 4.37±0.15pg / mL, 84.37±2.63pg / mL and 497.05±10.33pg / mL, respectively, and the recovery rates were between 84% and 99%, indicating that the method of the present invention can be used for the detection of aflatoxin AFB1 in flour.

[0168] 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 an electrochemical immunosensor for detecting aflatoxin B1, characterized in that: The electrochemical immunosensor comprises a modified electrode, an electrochemical nanoprobe and an AFB1 antibody. The preparation method of the electrochemical nanoprobe comprises the following steps: S10, Zn 2+ The solution, the curcumin solution, and the 2-methylimidazole solution are mixed to obtain a mixed solution, the mixed solution is shaken, centrifuged to collect a precipitate, and then washed to obtain CCM@ZIF-8; S20, dispersing the CCM@ZIF-8 in a solvent, then adding Tris-hydrochloric acid buffer and dopamine solution and mixing, then centrifuging to collect the precipitate, washing, and obtaining an intermediate; S30, dispersing the intermediate in a solvent, then adding activated anti-mouse immunoglobulin G, incubating at 35-38° C. for 25-35 min, then shaking at 3-5° C. for 10-14 h, finally centrifuging and collecting the precipitate, washing, and obtaining an electrochemical nanoprobe; The preparation method of the modified electrode comprises the following steps: A1. Clean the gold electrode, then modify it with DTSSP, then drop the AFB1-BSA solution on the surface of the gold electrode modified with DTSSP, then incubate and clean it, and finally drop the BSA solution for sealing to obtain a modified electrode.

2. The method for preparing the electrochemical immunosensor for detecting aflatoxin B1 according to claim 1, characterized in that: In step S10: In the mixed solution, 0.2-0.4 mol of Zn was added for every 0.072 μg of curcumin. 2+ and 0.1 to 0.2 mol of 2-methylimidazole.

3. The method for preparing the electrochemical immunosensor for detecting aflatoxin B1 according to claim 1, characterized in that: Step S10 includes: The mixed solution was shaken at 35-38° C. for 25-35 min, and then centrifuged at 3-5° C. and 10,000-12,000 r / min for 10-20 min, the precipitate was collected, and the precipitate was washed with methanol for multiple times to obtain CCM@ZIF-8.

4. The method for preparing the electrochemical immunosensor for detecting aflatoxin B1 according to claim 1, characterized in that: The Zn 2+ Zn in solution 2+ The molar ratio of the dopamine in the dopamine solution is 0.2-0.4:0.1; and / or, In step S20, in the centrifugation step, the centrifugal speed is 10000-12000 r / min, the centrifugal time is 10-20 min, and the centrifugal temperature is 3-5°C.

5. The method for preparing the electrochemical immunosensor for detecting aflatoxin B1 according to claim 1, characterized in that: Every 0.2~0.4mol Zn 2+ Correspondingly, 0.02 mg of anti-mouse immunoglobulin G was added.

6. The method for preparing the electrochemical immunosensor for detecting aflatoxin B1 according to claim 1, characterized in that: Step A1 includes: The gold electrode was cleaned and then modified with DTSSP. The AFB1-BSA solution was then dropped on the surface of the gold electrode modified with DTSSP. The electrode was then incubated at 35-38°C for 2 h, washed with PBST for several times, and finally BSA solution was added thereto for sealing to obtain a modified electrode.

7. The method for preparing the electrochemical immunosensor for detecting aflatoxin B1 according to claim 6, characterized in that: The diameter of the gold electrode is 2 mm; The concentration of the AFB1-BSA solution is 4-6 μg / mL, and the amount of each gold electrode added is 2-4 μL; and / or, The concentration of the AFB1 antibody was 5.0 μg / mL.

8. A method for detecting aflatoxin B1, characterized in that: The following steps are involved: Providing an electrochemical immunosensor, wherein the electrochemical immunosensor is prepared by the preparation method of the electrochemical immunosensor for detecting aflatoxin B1 according to any one of claims 1 to 7; After mixing the AFB1 antibody and the substance to be tested, the mixture is added dropwise to the surface of the modified electrode, incubated at 35-38°C for 50-70 minutes, and then washed with PBST for several times to obtain a first electrode; Dispersing the electrochemical nanoprobe in methanol to obtain a probe solution, dropping the probe solution onto the first electrode, incubating at 35-38° C. for 90-120 min, and then washing with PBST to obtain an electrode to be tested; The electrode to be tested is used as the working electrode, the saturated mercury electrode is used as the reference electrode, and the platinum wire electrode is used as the counter electrode, and the corresponding current value is detected; The AFB1 content in the substance to be tested is calculated according to the current value.

9. The method for detecting aflatoxin B1 according to claim 8, characterized in that: Add 2 to 4 μL of the AFB1 antibody to each modified electrode; and / or, 3 to 4 μL of the probe solution is dripped onto each of the first electrodes.

10. The method for detecting aflatoxin B1 according to claim 8, characterized in that: The substances to be tested include food and its products.

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