A multifunctional portable electrochemical method for detecting mycotoxins

Abstract

The present application belongs to the technical field of rapid detection of food and agricultural products, and particularly relates to a method for detecting mycotoxins by using multifunctional portable electrochemical technology. First, tetrahedral DNA specifically recognizing ochratoxin A and tetrahedral DNA specifically recognizing aflatoxin B1 are prepared, and then mixed and modified on a gold nanoflower screen-printed electrode to obtain an aptamer functionalized electrode. Then, the methylene blue@covalent organic framework modified by the aflatoxin B1 aptamer and the ferrocene@covalent organic framework modified by the ochratoxin A aptamer are mixed and modified on the surface of the aptamer functionalized electrode, and finally a multifunctional electrochemical sensor is obtained. The electrochemical sensor can be connected with a portable electrochemical workstation to form a portable electrochemical detection device, which can realize on-site collection and processing of electrochemical signals, and further realize rapid qualitative identification and quantitative detection of ochratoxin A and aflatoxin B1 in food, and is easy to operate and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of rapid detection of food and agricultural products, and particularly relates to a method for detecting mycotoxins by using a multifunctional portable electrochemical technique. BACKGROUND

[0002] Mycotoxins are low-molecular-weight toxic secondary metabolites produced by fungi, and have the characteristics of low concentration, high toxicity and coexistence of multiple toxins. Crops such as grains, oils and fruits are easily contaminated by mycotoxins during planting, growth, harvesting, transportation, storage and processing. The contamination of food by mycotoxins not only causes huge economic losses, but also causes great harm to animal and human health and a series of food safety and health problems once the contaminated food enters the food chain. Traditional detection methods for mycotoxins such as high-performance liquid chromatography and immunodetection have problems such as complex sample pretreatment, long detection time and expensive equipment.

[0003] In recent years, mycotoxin rapid detection methods based on nanosensing technology (fluorescence method, Raman method, electrochemical method, etc.) have attracted much attention due to their low detection cost and simple operation. Among them, electrochemical method has become a research hotspot due to its high sensitivity and strong selectivity. A series of electrochemical sensors have been developed to realize high-precision detection of aflatoxins, ochratoxin and fumonisin. However, the current electrochemical sensors for mycotoxin detection still have the following shortcomings: first, the existing electrochemical sensors are mostly for high-sensitivity and high-selectivity detection of single toxin, and the detection efficiency of complex food systems coexisting with multiple mycotoxins is low; second, mycotoxin detection still needs to rely on laboratory equipment, and cannot realize portable on-site detection, which limits the practical application of electrochemical sensors. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the application provides a method for detecting mycotoxins by using a multifunctional portable electrochemical technique. The multifunctional electrochemical sensor can realize rapid qualitative identification and quantitative detection of ochratoxin A and aflatoxin B1 in food. The portable acquisition device can realize on-site acquisition and processing of electrochemical signals. The method has the advantages of high detection efficiency, low cost and realization of multi-scene application.

[0005] To achieve the above-mentioned application purposes, the specific technical solutions of the application are as follows:

[0006] A method for detecting mycotoxins by using a multifunctional portable electrochemical technique mainly includes preparation of a multifunctional electrochemical sensor, establishment of a portable electrochemical signal acquisition method and portable rapid detection of mycotoxins.

[0007] I. Preparation of a multifunctional electrochemical sensor

[0008] S1. Covalent organic framework: 2,5-dimethoxybenzaldehyde and 1,3,5-tris(4- aminophenyl)benzene are dissolved in acetonitrile, ultrasonic oscillation is used for mixing, then glacial acetic acid is added, and the first stirring is carried out under certain temperature conditions. After stirring, benzaldehyde is added and the reaction is continued. After the reaction, centrifugation is carried out, and the product after centrifugation is collected and washed with ultrapure water, ethanol and methanol several times, and then vacuum dried to obtain the purified covalent organic framework;

[0009] The purified covalent organic framework is added to methylene blue solution and ferrocene solution respectively, and the second stirring is carried out. After the stirring reaction, methylene blue mixed solution and ferrocene mixed solution are obtained. Then, the two kinds of mixed solutions are centrifuged respectively, and the products after centrifugation are collected and washed with ultrapure water, ethanol and methanol several times, and then vacuum dried to obtain the purified methylene blue covalent organic framework and the purified ferrocene covalent organic framework;

[0010] Preferably, in S1 of step one, the amounts of 2,5-dimethoxybenzaldehyde, 1,3,5-tris(4- aminophenyl)benzene, acetonitrile, glacial acetic acid and benzaldehyde are 1-3 mg, 1-3 mg, 20-40 mL, 1-3 mL and 0.5-2 mL respectively.

[0011] The temperature for the first stirring under certain temperature conditions is 25-30℃, and the time is 2-5 hours. The time for the second stirring after adding benzaldehyde is 2-4 hours. The number of washing times is 3-5 times.

[0012] The amounts of the covalent organic framework and methylene blue solution are 5-15 mg and 10-20 mL respectively, and the concentration of the methylene blue solution is 1-2 mg / mL. The amounts of the covalent organic framework and ferrocene solution are 5-15 mg and 10-20 mL respectively, and the concentration of the ferrocene solution is 1-2 mg / mL.

[0013] S2. Ochratoxin A aptamer modified ferrocene covalent organic framework: Ochratoxin A aptamer (DNAa) solution is mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and deionized water, and the first stirring incubation is carried out at room temperature. Then, the purified ferrocene covalent organic framework in step S1 is added, and the second incubation is carried out at room temperature. Then, centrifugation is carried out, and the solid precipitate obtained is the ochratoxin A aptamer modified ferrocene covalent organic framework;

[0014] Preferably, the amount of the ochratoxin A aptamer solution, deionized water, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and purified ferrocene@covalent organic framework in step one S2 is 10-50 μL: 10-20 mL: 3-5 mg: 3-5 mg: 3-5 mg, wherein the concentration of the ochratoxin A aptamer solution is 10-50 μmol / L;

[0015] The first stirring incubation time is 2-4 hours; the second incubation time is 12-48 hours.

[0016] S3. Aflatoxin B1 aptamer modified methylene blue@covalent organic framework: mix the aflatoxin B1 aptamer (DNA b) solution with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and deionized water to obtain a mixed solution, then add the purified methylene blue@covalent organic framework in step S1 after the first stirring incubation at room temperature, and then perform the second incubation at room temperature, and then centrifuge the mixed solution to obtain the solid precipitate, which is the aflatoxin B1 aptamer modified methylene blue@covalent organic framework;

[0017] Preferably, the amount of the aflatoxin B1 aptamer solution, deionized water solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and purified ferrocene@covalent organic framework in step one S3 is 10-50 μL: 10-20 mL: 3-5 mg: 3-5 mg: 3-5 mg, wherein the concentration of the aflatoxin B1 aptamer solution is 10-50 μmol / L;

[0018] The first stirring incubation time is 2-4 hours; the second incubation time is 12-48 hours.

[0019] S4. Gold nanoflower screen-printed electrode: place the screen-printed electrode in a mixed solution of chloroauric acid and sodium sulfate for electroplating, then take out the screen-printed electrode and wash it with deionized water to obtain the gold nanoflower screen-printed electrode;

[0020] Preferably, the concentration of chloroauric acid in the mixed solution in step one S4 is 0.25-0.5 mol / L, and the concentration of sodium sulfate in the mixed solution is 60-80 mmol / L; the electroplating is performed at a potential of -0.6 V for 400-600 s.

[0021] S5. Preparation of specific tetrahedral DNA:

[0022] (a) respectively take three kinds of aptamer (sequence 1, sequence 2 and sequence 3) solution, recorded as aptamer A solution, aptamer B solution and aptamer C solution, then three kinds of aptamer solution and ochratoxin A complementary chain aptamer (sequence 4) solution mixed with TCEP solution and TM buffer solution, mixed solution after heating in water bath, after heating and placed in ice water and quickly cooled to 0~4 DEG C, and under the condition of 0~4 DEG C after standing get ochratoxin A specific recognition tetrahedral DNA, recorded as TDNa;

[0023] (b) respectively take three kinds of aptamer (sequence 1, sequence 2 and sequence 3) solution, recorded as aptamer A solution, aptamer B solution and aptamer C solution, then three kinds of aptamer solution and ochratoxin A complementary chain aptamer (sequence 4) solution mixed with TCEP solution and TM buffer solution, mixed solution after heating in water bath, after heating and placed in ice water and quickly cooled to 0~4 DEG C, and under the condition of 0~4 DEG C after standing get ochratoxin A specific recognition tetrahedral DNA, recorded as TDNa;

[0024] Preferably, the S5 of step one (a) in the aptamer A solution: aptamer B solution: aptamer C solution: ochratoxin A complementary chain aptamer solution: TCEP solution: TM buffer solution is 1-4 μL: 1-4 μL: 1-4 μL: 1-4 μL: 5-20 μL: 41-164 μL; the water bath heating temperature is 80-100 min, the time is 5-20 min;

[0025] The S5 of step one (b) in the aptamer A solution: aptamer B solution: aptamer C solution: ochratoxin A complementary chain aptamer solution: TCEP solution: TM buffer solution is 1-4 μL: 1-4 μL: 1-4 μL: 1-4 μL: 5-20 μL: 41-164 μL; the water bath heating temperature is 80-100 min, the time is 5-20 min;

[0026] Preferably, the three kinds of aptamer mentioned in (a) and (b) in step one S5 are the same substance, and the volume ratio of aptamer A solution, aptamer B solution, aptamer C solution, ochratoxin A complementary chain aptamer solution and yellow aspergillus toxin B1 complementary chain aptamer solution is 1:1:1:1:1; the standing time is 10-30 min;

[0027] The concentration of the aptamer A solution is 10-50 μmol / L, the concentration of the aptamer B solution is 10-50 μmol / L, the concentration of the aptamer C solution is 10-50 μmol / L, the concentration of the ochratoxin A complementary strand aptamer solution is 10-50 μmol / L, and the concentration of the aflatoxin B1 complementary strand aptamer solution is 10-50 μmol / L.

[0028] S6. Aptamer-functionalized gold nanoflower screen-printed electrode: the TDNa and TDNb prepared in S5 are mixed and then dropped on the gold nanoflower screen-printed electrode in S4, and after incubation at room temperature, the electrode is washed with deionized water to obtain an aptamer-functionalized electrode;

[0029] Preferably, the volume ratio of TDNa and TDNb in S6 of step one is 1:1, and the dropping amount is 50-350 μL; the incubation time is 5-10 hours.

[0030] S7. Multifunctional electrochemical sensor assembly: the aflatoxin B1 aptamer modified methylene blue@covalent organic framework in S3 is added into ultrapure water to obtain a mixed solution, which is denoted as solution A;

[0031] The ochratoxin A aptamer modified ferrocene@covalent organic framework in S2 is added into ultrapure water to obtain a mixed solution, which is denoted as solution B;

[0032] After mixing solution A and solution B, they are dropped on the surface of the aptamer-functionalized electrode in S6, and after incubation at room temperature, the multifunctional electrochemical sensor is obtained after washing with deionized water;

[0033] Preferably, the concentration of solution A and solution B in S7 of step one is 0.1-1 mg / ml; the volume ratio of the mixed solution A and solution B is 1:1, the dropping amount is 200 μL, and the incubation time is 10-15 hours.

[0034] II. Portable acquisition method of electrical signals;

[0035] S1. Assembly of portable electrochemical workstation: the multifunctional electrochemical sensor is connected to the electrochemical workstation through a screen-printed electrode adapter to obtain a portable electrochemical workstation for acquiring electrochemical signals;

[0036] S2. Fungus toxin incubation and electrical signal acquisition stage: mix aflatoxin B1 and ochratoxin A to prepare standard solutions with different concentrations; the concentration of aflatoxin B1 is denoted as B1, B2, …, Bn-1, Bn, n is a positive integer; the concentration of ochratoxin A is denoted as A1, A2, …, An-1, An, n is a positive integer; then drop the above standard solutions on the electrode of the multifunctional electrochemical sensor of the portable electrochemical workstation described in S1 of step (2) to perform incubation, after the incubation is completed, flush the electrode with deionized water again to obtain the incubated multifunctional electrochemical sensor; and drop electrolyte on the electrode surface of the incubated multifunctional electrochemical sensor, then use the portable electrochemical workstation described in S1 of step (2) to collect electrochemical signals, measure and record the electrical signal intensity of the aflatoxin B1 and ochratoxin A mixture standard sample with different concentrations, wherein the electrical signal intensity of the ochratoxin A standard sample is denoted as a1, a2, …, an-1, an, n is a positive integer; the electrical signal intensity of the aflatoxin B1 standard sample is denoted as b1, b2, …, bn-1, bn, n is a positive integer;

[0037] Preferably, the amount of standard sample dropped in S2 of step two is 200-300 μL, the incubation time is 1-2 h, and the deionized water is flushed 3-5 times; the concentration of aflatoxin B1 in the standard solution is 0.1-60 μg / L, and the concentration of ochratoxin A is 0.1-60 μg / L; the electrolyte is phosphate electrolyte with a concentration of 5-15 mmol / L and a pH of 7.2, and the amount of electrolyte dropped is 200-300 μL; the potential scanning range is -0.5 V-0.2 V, and the recorded electrical signal intensity is measured three times to take the average.

[0038] S3. Rapid prediction model of fungus toxin concentration;

[0039] (a) Rapid detection prediction model of ochratoxin A: according to the electrical signal intensity a1, a2, …, an-1, an of ochratoxin A described in S2 of step (2) and the ochratoxin A standard sample A1, A2, …, An-1, An with different concentrations, a rapid detection prediction model a=f(x) of ochratoxin A is established, wherein a is the oxidation peak intensity of the electrode, x is the concentration of ochratoxin A, and n is a positive integer;

[0040] (b) Rapid detection prediction model of aflatoxin B1: according to the electrical signal intensity b1, b2, …, bn-1, bn of aflatoxin B1 described in S2 of step (2) and the aflatoxin B1 standard sample B1, B2, …, Bn-1, Bn with different concentrations, a rapid detection prediction model b=f(y) of aflatoxin B1 is established, wherein b is the oxidation peak intensity of the electrode, y is the concentration of aflatoxin B1, and n is a positive integer.

[0041] III. Portable rapid detection of mycotoxins

[0042] The test substance is added dropwise to the electrode surface of the multifunctional electrochemical sensor described in step one S7, incubated, then washed with deionized water, and the electrolyte is added dropwise to the electrode surface of the incubated multifunctional electrochemical sensor, then the portable electrochemical workstation described in step two S1 is used to collect the electrochemical signal, and the corresponding electrical signal intensity of ochratoxin A is a; the corresponding electrical signal intensity of aflatoxin B1 is b; the detection prediction model in step two S3 is used to realize the detection of ochratoxin A and aflatoxin B1 in the test substance.

[0043] Preferably, the amount of the test substance added dropwise in step three is 200-300 μL, the incubation time is 2-3 hours, the number of deionized water washing is 3-5 times, the electrolyte is phosphate electrolyte, the concentration is 5-15 mmol / L, the pH is 7.2, and the dropwise amount is 200-300 μL.

[0044] Advantages of the present application

[0045] The present application proposes a multifunctional electrochemical sensor for detecting mycotoxins in food, which can simultaneously identify ochratoxin A and aflatoxin B1 in the test sample, and simultaneously realize qualitative identification and quantitative detection of the two toxins, greatly improving the detection efficiency of mycotoxins and the utilization rate of the sensor.

[0046] The present application proposes the use of tetrahedral DNA connection electrodes and single-stranded DNA, which can not only specifically identify mycotoxins, but also control the distribution, concentration and direction of aptamers on the electrode surface, greatly improving the stability and accuracy of nanosensors in complex systems.

[0047] The multifunctional portable electrochemical detection method for mycotoxins proposed in the present application uses a portable signal acquisition device and a highly integrated electrochemical sensor, which can be used in different detection scenarios and can realize on-site rapid detection of mycotoxins.

[0048] The multifunctional portable electrochemical detection method for mycotoxins proposed in the present application has a detection level of ochratoxin A and aflatoxin B1 lower than the national standard, meeting the needs of daily detection. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 Response of multifunctional electrochemical sensor to ochratoxin A.

[0050] Figure 2 Response of multifunctional electrochemical sensor to aflatoxin B1. DETAILED DESCRIPTION

[0051] The application will be described in detail below by various embodiments. However, these embodiments are only used to illustrate the application and not to limit the scope of the application.

[0052] In addition, after reading the content described in the application, those skilled in the art can make various modifications or changes to the application, and these equivalent forms also fall within the scope defined by the claims attached to the application.

[0053] The screen-printed electrode used in the application is purchased from Wave Probe Technology (Weihai) Co., Ltd., and the portable electrochemical workstation is purchased from Haoyang Technology Co., Ltd.

[0054] The aptamer A, the aptamer B, the aptamer C, the ochratoxin A complementary chain aptamer and the aflatoxin B1 complementary chain aptamer are all purchased from Shengong Bioengineering (Shanghai) Co., Ltd.; the aptamer sequences involved in the application are all conventional reagent primers, and the application does not involve a sequence listing.

[0055] The aptamer sequences involved in the application are shown in Table 1:

[0056] Table 1: Aptamer sequences involved in the application

[0057]

[0058] Example 1:

[0059] A multifunctional portable electrochemical technology for detecting fungal toxins, and the specific steps are as follows:

[0060] I. Preparation of a multifunctional electrochemical sensor;

[0061] S1. Covalent organic framework:

[0062] 2mg of 2,5-dimethoxybenzaldehyde and 2mg of 1,3,5-tris(4-aminophenyl)benzene were dissolved in 20mL of acetonitrile, ultrasonically mixed, 1mL of glacial acetic acid was added, and stirring was carried out at 25℃ for 4 hours, 1mL of benzaldehyde was added and stirring was continued for 2 hours. After the reaction, the product was washed with ultrapure water, ethanol and methanol in sequence for 3 times, and dried in a vacuum drying box to obtain a purified covalent organic framework; 10mg of the covalent organic framework was taken and mixed with 1mg / mL methylene blue (10mL); at the same time, another 10mg of the covalent organic framework was taken and mixed with 1mg / mL ferrocene (10mL) for 4 hours, and after the reaction, the products were washed with ultrapure water, ethanol and methanol in sequence for 3 times, and dried in a vacuum drying box to obtain purified methylene blue@covalent organic framework and ferrocene@covalent organic framework;

[0063] S2. Ochratoxin A aptamer modified ferrocene@covalent organic framework:

[0064] Mix 50 pmol / L of ochratoxin A aptamer (DNAa) solution (20 μL) with 2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 2 mg of N-hydroxysuccinimide (NHS) in 10 mL of deionized water, add the purified ferrocene@covalent organic framework (3 mg) in step S1 after stirring at room temperature for 3 hours, incubate at room temperature for 24 hours, then centrifuge the product, and the solid precipitate obtained is the ochratoxin A aptamer modified ferrocene@covalent organic framework;

[0065] S3. aflatoxin B1 aptamer modified methylene blue@covalent organic framework:

[0066] Mix 50 pmol / L of aflatoxin B1 aptamer (DNAb) solution (20 μL) with 2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 2 mg of N-hydroxysuccinimide (NHS) in 10 mL of deionized water, add the purified methylene blue@covalent organic framework (3 mg) in step S1 after stirring at room temperature for 3 hours, incubate at room temperature for 24 hours, then centrifuge the product, and the solid precipitate obtained is the aflatoxin B1 aptamer modified methylene blue@covalent organic framework;

[0067] S4. gold nanoflower screen-printed electrode:

[0068] Place the screen-printed electrode in a mixed solution (containing 0.25 mol / L chloroauric acid and 60 mmol / L sodium sulfate) and electroplate at a potential of -0.6 V for 400 s, rinse with deionized water for 2 times, and obtain the gold nanoflower screen-printed electrode;

[0069] S5. preparation of specific tetrahedral DNA:

[0070] Take 2 μL of each of 50 pmol / L of three aptamers (sequence 1, sequence 2 and sequence 3) solution and 50 pmol / L of ochratoxin A complementary strand aptamer (sequence 4) solution into a centrifuge tube, add 20 μL of TCEP solution and 164 μL of TM buffer at the same time, mix well. Place the centrifuge tube in 90℃ water and heat for 10 min, immediately take out and place in ice water to quickly cool to 4℃, and stand in a 4℃ refrigerator for 30 min, to obtain the ochratoxin A specific recognition tetrahedral DNA (TDNa).

[0071] Take 2 μL of 50 μmol / L aptamer (sequence 1, sequence 2 and sequence 3) solution and 50 μmol / L aflatoxin B1 complementary strand aptamer (sequence 4) solution respectively into a centrifugal tube, add 20 μL of TCEP solution and 164 μL of TM buffer solution at the same time, and mix well. Place the centrifugal tube in 90°C water for heating for 10 min, immediately take it out and place it in ice water for rapid cooling to 4°C, and place it in a 4°C refrigerator for 30 min to obtain tetrahedral DNA (TDNb) specifically recognized by aflatoxin B1.

[0072] S6. Gold nanoflower screen-printed electrode functionalized by aptamer:

[0073] Mix the tetrahedral DNA solution specifically recognized by aflatoxin B1 and ochratoxin A 1:1 and drop it on the gold nanoflower screen-printed electrode described in S4. After incubation at room temperature for 5 hours, wash the surface of the electrode with deionized water to remove the unbound tetrahedral DNA, and obtain an electrode functionalized by aptamer;

[0074] S7. Assembly of multifunctional electrochemical sensor:

[0075] Mix 0.2 mg / mL of methylene blue@covalent organic framework modified by aflatoxin B1 aptamer and 0.2 mg / mL of ferrocene@covalent organic framework modified by ochratoxin A aptamer according to a volume ratio of 1:1, and take 200 μL to drop on the surface of the electrode functionalized by aptamer described above. After incubation at room temperature for 10 hours, wash the surface of the electrode with deionized water to remove the unbound substances, and obtain a multifunctional electrochemical sensor.

[0076] II. Acquisition of electrical signal by portable acquisition method

[0077] S1. Assembly of portable electrochemical workstation: connect the electrochemical workstation to the mobile phone and electrode adapter through USB data line and electrode line respectively; connect the multifunctional electrochemical sensor in S7 of step 1 above to the electrochemical workstation through the electrode adapter to obtain a portable electrochemical workstation;

[0078] S2. Fungal toxin incubation: take 250 μL of different concentrations of aflatoxin B1 and ochratoxin A mixture standard sample and drop it on the electrode surface of the multifunctional electrochemical sensor of the portable electrochemical workstation described in S1 of step 2; incubate for 3 hours. In this process, the ferrocene@covalent organic framework (DNAa@FcCOF) modified by ochratoxin A aptamer and the methylene blue@covalent organic framework (DNAb@MBCOF) modified by aflatoxin B1 aptamer are detached from the multifunctional electrochemical sensor. Wash the electrode surface of the multifunctional electrochemical sensor with deionized water for 3 times for detection;

[0079] S3. The electrical signal acquisition stage: 200 μL of 10 mmol / L phosphate buffer solution was added to the electrode surface of the multifunctional electrochemical sensor, and the multifunctional electrochemical sensor prepared in step (2) S2 was subjected to potential scanning, with the potential being scanned from -0.6 V to 0.2 V, to obtain the electrical signal of the electrode corresponding to different concentrations of mycotoxins. The electrical signal intensity of the mixture standard sample of aflatoxin B1 and ochratoxin A at different concentrations was measured and recorded, wherein the electrical signal intensity of the ochratoxin A standard sample (as shown in Figure 1 ) was 105, 94, 84, 80, 76, 67, 59, 53, 42, 38, 30, 28, 25 mA, respectively; and the electrical signal intensity of the aflatoxin B1 standard sample (as shown in Figure 2 ) was 102, 93, 85, 79, 76, 70, 63, 52, 45, 36, 30, 28, 24 mA, respectively.

[0080] S5. Rapid prediction model of mycotoxin concentration

[0081] Rapid detection prediction model of ochratoxin A: a model was established according to the electrical signal intensity 105, 94, 84, 80, 76, 67, 59, 53, 42, 38, 30, 28, 25 of ochratoxin A and different concentrations of ochratoxin A standard sample 0.1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 μg / L in step (2) S3, to obtain the rapid detection prediction model of ochratoxin A a = -1.4080x + 101.73, R 2 = 0.9916, wherein a is the oxidation peak intensity of the electrode, and x is the concentration of ochratoxin A.

[0082] Rapid detection prediction model of aflatoxin B1: a model was established according to the electrical signal intensity 102, 93, 85, 79, 76, 70, 63, 52, 45, 36, 30, 28, 24 of aflatoxin B1 and different concentrations of aflatoxin B1 standard sample 0.1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 μg / L in step (2) S3, to obtain the rapid detection prediction model of aflatoxin B1 b = -1.3437y + 100.55, R 2 = 0.9899, wherein b is the oxidation peak intensity of the electrode, and y is the concentration of aflatoxin B1.

[0083] III. Specific detection is carried out by taking peanuts as an example.

[0084] Rapid prediction of the content of aflatoxin B1 or ochratoxin A in peanuts:

[0085] Take 5 portions of peanut samples to be tested for natural mold growth, each 20±1g, crush the samples with a pulverizer, sieve, accurately weigh 5g (accurate to 0.01g) of the sample into a 50mL centrifuge tube, add 20mL of acetonitrile-water-acetic acid mixture (70:29:1, by volume), shake or vortex extraction for 30min, then centrifuge at 6000r / min for 10min, aspirate 0.5mL of supernatant into a 1.5mL centrifuge tube, add 0.5mL of water and vortex well, centrifuge at 12000r / min for 10min at 4℃, pass the supernatant through a 0.2μm polytetrafluoroethylene filter membrane, collect the filtrate as the test solution.

[0086] Drop 200μL of the test solution on the electrode surface of the multifunctional electrochemical sensor, incubate at room temperature for 3 hours, rinse the electrode surface with ultrapure water to remove unreacted substances and substances falling off from the electrode surface, then add 200μL of phosphate buffer on the electrode surface, obtain the electrode electrical signal in the range of-0.6~0.2V, repeat the measurement three times; the corresponding electrical signal intensity of aflatoxin A is a, and the corresponding electrical signal intensity of aflatoxin B1 is b;

[0087] Substitute the electrical signal intensity b into the rapid prediction model of aflatoxin B1 to obtain the concentration of aflatoxin B1, and the obtained aflatoxin B1 is 0.462μg / kg, 1.068μg / kg, 8.526μg / kg, 15.41μg / kg, 24.65μg / kg, respectively.

[0088] In order to verify the detection accuracy of the method described in the application, the test solutions of the 5 groups of samples were detected according to the national food safety standard (GB 5009.22-2016), and the results of aflatoxin B1 were 0.465μg / kg, 1.062μg / kg, 8.519μg / kg, 15.42μg / kg, 24.67μg / kg, respectively. The relative error of the detection results of the method proposed in the application is less than 1%, indicating that the multifunctional portable electrochemical detection method of fungal toxins described in the application has good practicability for the detection of aflatoxin B1 in food samples.

[0089] The concentration of ochratoxin A is obtained by substituting the electric signal intensity a into the rapid prediction model of ochratoxin A, and the obtained ochratoxin A is 2.358 μg / kg, 8.579 μg / kg, 14.25 μg / kg, 26.34 μg / kg and 38.56 μg / kg respectively. Similarly, the five groups of samples are detected according to the national food safety standard (GB 5009.96-2016), and the obtained ochratoxin A results are 2.361 μg / kg, 8.582 μg / kg, 14.24 μg / kg, 26.36 μg / kg and 38.54 μg / kg respectively. The relative error of the detection results of the method proposed in the application is less than 1%, indicating that the multifunctional portable electrochemical detection method of the fungal toxin has good practicability for detecting ochratoxin A in food samples.

[0090] In summary, the multifunctional electrochemical sensor for detecting fungal toxins in food is proposed in the application, which can simultaneously identify ochratoxin A and aflatoxin B1 in the sample to be detected, and simultaneously realize qualitative identification and quantitative detection of the two toxins, greatly improving the detection efficiency of fungal toxins and the utilization rate of the sensor; the detection level of ochratoxin A and aflatoxin B1 is lower than the national standard, meeting the needs of daily detection.

[0091] It is to be noted that the above examples are only used to illustrate the technical solutions described in the application and not to limit the application; therefore, although the application has been described in detail with reference to the above examples, those skilled in the art should understand that the application can still be modified or replaced equivalently; and all technical solutions and improvements without departing from the spirit and scope of the application should be covered in the scope of the claims of the application.

Claims

1. A method for preparing a multifunctional electrochemical sensor for detecting mycotoxins, characterized in that, Includes the following steps: S1. Dissolve 2,5-dimethoxybenzaldehyde and 1,3,5-tris(4-aminophenyl)benzene in acetonitrile, mix by ultrasonic vibration, then add glacial acetic acid and stir for the first time under a certain temperature condition. After stirring, add benzaldehyde and continue stirring to react. After the reaction, centrifuge and collect the product after centrifugation. Wash the product several times with ultrapure water, ethanol and methanol in sequence. After washing, dry under vacuum to obtain the purified covalent organic framework. The purified covalent organic framework was added to methylene blue solution and ferrocene solution respectively, and stirred for a second time. After stirring, a methylene blue mixture and a ferrocene mixture were obtained. The two mixtures were then centrifuged separately, and the centrifuged products were collected and washed several times with ultrapure water, ethanol and methanol in sequence. After vacuum drying, purified methylene blue@covalent organic framework and ferrocene@covalent organic framework were obtained. S2. The ochratoxin A aptamer solution was mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and deionized water. After the first stirring incubation at room temperature, the ferrocene@covalent organic framework purified in step S1 was added. After the second incubation at room temperature, the mixture was centrifuged. The resulting solid precipitate was the ochratoxin A aptamer-modified ferrocene@covalent organic framework. S3. The aflatoxin B1 aptamer solution was mixed with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and deionized water. After the mixed solution was stirred and incubated for the first time at room temperature, the purified methylene blue@covalent organic framework from step S1 was added. After a second incubation at room temperature, the mixed solution was centrifuged. The solid precipitate obtained was the aflatoxin B1 aptamer modified with methylene blue@covalent organic framework. S4. Placing the screen-printed electrode in a mixed solution of chloroauric acid and sodium sulfate for electroplating, and then rinsing the screen-printed electrode with deionized water to obtain a gold nanoflower screen-printed electrode. S5. Preparation of specific tetrahedral DNA: (a) Take three aptamer solutions, denoted as aptamer A solution, aptamer B solution and aptamer C solution respectively. Then mix the three aptamer solutions with the ochratoxin A complementary strand aptamer solution, add TCEP solution and TM buffer at the same time, mix well and place the mixture in a water bath for heating. After heating, remove it and place it in ice water for rapid cooling to 0~4℃. After standing at 0~4℃, the tetrahedral DNA specifically recognized by ochratoxin A is obtained, denoted as TDNa. (b) Take three aptamer solutions, denoted as aptamer A solution, aptamer B solution and aptamer C solution respectively. Then mix the three aptamer solutions with the aflatoxin B1 complementary strand aptamer solution, add TCEP solution and TM buffer at the same time, mix well and place the mixture in a water bath for heating. After heating, remove it and place it in ice water for rapid cooling to 0~4℃. After standing at 0~4℃, the tetrahedral DNA specifically recognized by aflatoxin B1 is obtained, denoted as TDNb. The sequence of aptamer A is: SH-C6 TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGATGC GAG GGT CCA ATA C; The sequence of aptamer B is: SH-C6 TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCTACT ATG GCG GCT CTT C; The sequence of aptamer C is: SH-C6 TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTGTAT TGG ACC CTC GCA T; S6. The TDNa and TDNb prepared in S5 are mixed and dropped onto the gold nanoflower silk screen printed electrode described in S4. After incubation at room temperature, the electrode is rinsed with deionized water to obtain the aptamer-functionalized electrode. S7. Add the methylene blue@covalent organic framework modified with the aflatoxin B1 aptamer from S3 to ultrapure water to obtain a mixed solution, denoted as solution A; Ferrocene@covalent organic framework modified with ochratoxin A aptamer in S2 was added to ultrapure water to obtain a mixed solution, denoted as solution B. After mixing solutions A and B, the mixture was dropped onto the surface of an electrode functionalized with S6 aptamer. After incubation at room temperature and rinsing with deionized water, a multifunctional electrochemical sensor was obtained.

2. The method for preparing the multifunctional electrochemical sensor for detecting mycotoxins according to claim 1, characterized in that, In step S1, the amounts of 2,5-dimethoxybenzaldehyde, 1,3,5-tris(4-aminophenyl)benzene, acetonitrile, glacial acetic acid, and benzaldehyde used are 1~3 mg: 1~3 mg: 20~40 mL: 1~3 mL: 0.5~2 mL, respectively. The temperature for the first stirring under the specified conditions is 25~30 °C, and the time is 2~5 hours; the time for adding benzaldehyde and continuing the stirring reaction is 2~4 hours; the number of washing cycles is 3~5 times. The amounts of the covalent organic framework and the methylene blue solution used are 5~15 mg: 10~20 mL, wherein the concentration of the methylene blue solution is 1~2 mg / mL; the amounts of the covalent organic framework and the ferrocene solution used are 5~15 mg: 10~20 mL, wherein the concentration of the ferrocene solution is 1~2 mg / mL.

3. The method for preparing the multifunctional electrochemical sensor for detecting mycotoxins according to claim 1, characterized in that, In step S2, the amounts of ochratoxin A aptamer solution, deionized water, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and purified ferrocene@covalent organic framework are in the following ratio: 10-50 μL: 10-20 mL: 3-5 mg: 3-5 mg: 3-5 mg, wherein the concentration of the ochratoxin A aptamer solution is 10-50 μmol / L. The first stirring and incubation period is 2-4 hours; the second incubation period is 12-48 hours.

4. The method for preparing the multifunctional electrochemical sensor for detecting mycotoxins according to claim 1, characterized in that, In step S3, the amounts of aflatoxin B1 aptamer solution, deionized water solution, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and purified ferrocene@covalent organic framework are in the following ratio: 10-50 μL: 10-20 mL: 3-5 mg: 3-5 mg: 3-5 mg, wherein the concentration of the aflatoxin B1 aptamer solution is 10-50 μmol / L. The first stirring and incubation period is 2-4 hours; the second incubation period is 12-48 hours.

5. The method for preparing the multifunctional electrochemical sensor for detecting mycotoxins according to claim 1, characterized in that, In step S4, the concentration of chloroauric acid in the mixed solution is 0.25~0.5 mol / L, and the concentration of sodium sulfate in the mixed solution is 60~80 mmol / L; the electroplating is performed at a potential of -0.6V for 400~600s.

6. The method for preparing the multifunctional electrochemical sensor for detecting mycotoxins according to claim 1, characterized in that, In step S5(a), the volumes of aptamer A solution, aptamer B solution, aptamer C solution, ochratoxin A complementary strand aptamer solution, TCEP solution, and TM buffer are 1~4 μL, 1~4 μL, 1~4 μL, 1~4 μL, 5~20 μL, and 41~164 μL, respectively; the water bath heating temperature is 80~100 min, and the time is 5~20 min. The volume of aptamer A solution, aptamer B solution, aptamer C solution, aflatoxin B1 complementary strand aptamer solution, TCEP solution, and TM buffer in step S5(b) is 1~4 μL: 1~4 μL: 1~4 μL: 1~4 μL: 5~20 μL: 41~164 μL; the water bath heating temperature is 80~100 min, and the time is 5~20 min; The three aptamers mentioned in steps S5 (a) and (b) are the same substances, and the volume ratio of aptamer A solution, aptamer B solution, aptamer C solution, ochratoxin A complementary chain aptamer solution and aflatoxin B1 complementary chain aptamer solution is 1:1:1:1:1; the standing time is 10-30 min for each of them. The concentrations of the aptamer A solution, aptamer B solution, aptamer C solution, ochratoxin A complementary strand aptamer solution, and aflatoxin B1 complementary strand aptamer solution are all 10-50 μmol / L.

7. The method for preparing the multifunctional electrochemical sensor for detecting mycotoxins according to claim 1, characterized in that, In step S6, the volume ratio of TDNa to TDNb is 1:1, and the amount added is 50-350 μL; the incubation time is 5-10 hours.

8. The method for preparing the multifunctional electrochemical sensor for detecting mycotoxins according to claim 1, characterized in that, In step S7, the concentrations of both solution A and solution B are 0.1-1 mg / ml; the volume ratio of solution A to solution B is 1:1, the amount added is 200 μL, and the incubation time is 10-15 hours.

9. The use of the multifunctional electrochemical sensor prepared according to any one of claims 1-8 for detecting mycotoxins, characterized in that, Includes the following steps: (1) Portable method for acquiring electrical signals; S1. Assembly of the portable electrochemical workstation: The multifunctional electrochemical sensor and the electrochemical workstation are connected by a screen-printed electrode adapter to obtain the portable electrochemical workstation in order to acquire electrochemical signals; S2. Mycotoxin incubation and electrical signal acquisition stage: Aflatoxin B1 and ochratoxin A are mixed to prepare standard solutions of different concentrations; the concentrations of aflatoxin B1 are denoted as B1, B2, ..., Bn-1, Bn, where n is a positive integer; the concentrations of ochratoxin A are denoted as A1, A2, ..., An-1, An, where n is a positive integer; then the above standard solutions are dropped onto the electrodes of the multifunctional electrochemical sensor of the portable electrochemical workstation described in S1 for incubation. After incubation, the electrodes are rinsed again with deionized water to obtain the incubated multifunctional electrochemical sensor; Electrolyte was added to the electrode surface of the multifunctional electrochemical sensor after incubation. Then, the electrochemical signal intensity of the mixed standard samples of aflatoxin B1 and ochratoxin A at different concentrations was collected and recorded using the portable electrochemical workstation described in S1. The electrochemical signal intensity of the ochratoxin A standard sample was denoted as a1, a2, ..., an-1, an, where n is a positive integer; the electrochemical signal intensity of the aflatoxin B1 standard sample was denoted as b1, b2, ..., bn-1, bn, where n is a positive integer. S3. A rapid predictive model for mycotoxin concentrations; (a) Rapid detection prediction model for ochratoxin A: Based on the electrical signal intensities a1, a2, ..., an-1, an of ochratoxin A described in S2 and standard samples A1, A2, ..., An-1, An of different concentrations of ochratoxin A, a model is established to obtain the rapid detection prediction model for ochratoxin A, a=f(x), where a is the oxidation peak intensity of the electrode, x is the concentration of ochratoxin A, and n is a positive integer; (b) Rapid detection prediction model for aflatoxin B1: The electrical signal intensities b1, b2, ..., bn-1, bn of aflatoxin B1 described in S2 are used to establish a model with standard samples B1, B2, ..., Bn-1, Bn of aflatoxin B1 at different concentrations to obtain a rapid detection prediction model for aflatoxin B1, b=f(y), where b is the oxidation peak intensity of the electrode, y is the concentration of aflatoxin B1, and n is a positive integer; (2) Portable rapid detection of fungal toxins; The analyte is dropped onto the electrode surface of a multifunctional electrochemical sensor, incubated, rinsed with deionized water, and then an electrolyte is dropped onto the electrode surface of the incubated multifunctional electrochemical sensor. The electrochemical signal is then collected using the portable electrochemical workstation described in S1 to obtain the electrical signal intensity a corresponding to ochratoxin A and the electrical signal intensity b corresponding to aflatoxin B1. The corresponding signals are then substituted into the detection prediction model in S3 to achieve the detection of ochratoxin A and aflatoxin B1 in the analyte.

10. The use according to claim 9, characterized in that, In step (1), the amount of standard sample added in S2 is 200-300 μL, the incubation time is 1-2 h, and the number of times to rinse with deionized water is 3-5; the concentration of aflatoxin B1 in the standard solution is 0.1-60 μg / L, and the concentration of ochratoxin A is 0.1-60 μg / L. The electrolyte was a phosphate electrolyte with a concentration of 5–15 mmol / L and a pH of 7.

2. The amount added was 200–300 μL. The potential scan range was -0.5 V to 0.2 V. The recorded electrical signal intensity was the average of three measurements.

11. The use according to claim 9, characterized in that, In step (2), the amount of analyte added is 200-300 μL, the incubation time is 2-3 hours, the number of times to rinse with deionized water is 3-5, the electrolyte is phosphate electrolyte with a concentration of 5-15 mmol / L and pH of 7.2, and the amount added is 200-300 μL.

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