Probe for detecting mycotoxin, preparation method and application thereof, and detection method for aflatoxin B1

Abstract

The present invention relates to the technical field of biosensing and nucleic acid detection, and discloses a probe for detecting mycotoxin, a preparation method and application thereof, and a method for detecting aflatoxin B1. The preparation method comprises the following steps: (1) Mixing a coupling agent I, an ETDA salt and water to carry out an activation reaction I to obtain an activation reaction solution I, and mixing the activation reaction solution I with a nucleic acid aptamer corresponding to a detection substance to carry out a contact reaction I to obtain a solution containing an EDTA salt@Apt probe; Mixing a coupling agent II, silica and water to carry out an activation reaction II to obtain an activation reaction solution II, and mixing the activation reaction solution II with a DNA complementary to the nucleic acid aptamer to carry out a contact reaction II, and obtaining a SiO2@DNA probe after solid-liquid separation I; (2) Mixing the EDTA salt@Apt probe, the SiO2@DNA probe and water to carry out a contact reaction III, and obtaining a probe after solid-liquid separation II. The probe has higher sensitivity and can effectively reduce the influence of environmental noise on detection.

Description

Technical Field

[0001] The present invention relates to the field of biosensing and nucleic acid detection technology, and in particular to a probe for detecting mycotoxins, a preparation method and an application thereof. In addition, the present invention also relates to a method for detecting aflatoxin B1 using the probe. Background Art

[0002] In recent years, food safety issues caused by mycotoxins have become a hot topic of great concern, and increasingly stringent limit standards require ultra-high sensitivity mycotoxin detection methods.

[0003] At present, the traditional mycotoxin detection methods include thin layer chromatography, liquid chromatography-mass spectrometry, high performance liquid chromatography, enzyme-linked immunosorbent assay, radioimmunoassay and immunochromatography, etc., and these methods have the disadvantages of insufficient sensitivity, low accuracy and poor repeatability, complex pre-treatment, strong professional operation, need for professional operators, long detection time and expensive instruments. The detection sensitivity (μg / mL-ng / mL) of the traditional enzyme-linked immunosorbent assay enzyme-catalyzed substrate is relatively low, and it is impossible to accurately quantitatively analyze some trace or even trace targets. The new fluorescent enzyme-linked immunosorbent assay technology uses enzyme-catalyzed organic luminescent substrates to generate fluorescence as a detection signal, but due to the low luminescence efficiency of traditional organic luminescent substrates, poor optical stability and susceptibility to environmental influences, its detection sensitivity is insufficient. At the same time, traditional fluorescent dyes have the disadvantage of fluorescence quenching in practical applications, and currently researchers are mostly focused on improving the intensity of fluorescent signals, while ignoring the influence of background noise on detection sensitivity. Moreover, the detection range and sensitivity of the probes used for detection in the prior art need to be improved. Summary of the invention

[0004] The purpose of the present invention is to overcome the problem of insufficient probe sensitivity in the prior art, and to provide a probe for detecting mycotoxins, a preparation method and application thereof, and a method for detecting aflatoxin B1. The probe has higher sensitivity and can effectively reduce the impact of environmental noise on detection.

[0005] In order to achieve the above object, the present invention provides a method for preparing a probe for detecting mycotoxins, comprising the following steps:

[0006] (1) mixing a coupling agent I, ETDA salt and water to perform an activation reaction I to obtain an activation reaction solution I, and mixing the activation reaction solution I with a nucleic acid aptamer corresponding to a detection substance to perform a contact reaction I to obtain a solution containing EDTA salt@Apt probe;

[0007] Couple coupling agent II, silica, and water and perform activation reaction II to obtain activation reaction solution II. Mix the activation reaction solution II with DNA complementary to the nucleic acid aptamer and perform contact reaction II. After solid-liquid separation I, obtain the SiO2@DNA probe;

[0008] (2) Mix the solution containing the EDTA salt@Apt probe, the SiO2@DNA probe, and water and perform contact reaction III. After solid-liquid separation II, obtain the probe.

[0009] Preferably, in step (1), the coupling agent I and the coupling agent II are N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, respectively.

[0010] More preferably, in the coupling agent I and the coupling agent II, the molar ratio of N-hydroxysuccinimide to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is independently 1:0.5-2.

[0011] Preferably, the EDTA salt is potassium EDTA and / or sodium EDTA.

[0012] Preferably, in step (1), the time of activation reaction I is 10-25 min, and the time of contact reaction I is 1-3 h;

[0013] The time of activation reaction II is 10-25 min, and the time of contact reaction II is 1-3 h.

[0014] Preferably, in step (2), the time of contact reaction III is 20-40 min.

[0015] Preferably, the molar ratio of the EDTA salt@Apt probe to the SiO2@DNA probe is 1:4-16.

[0016] Preferably, the detection substance is aflatoxin B1.

[0017] More preferably, the nucleotide sequence of the nucleic acid aptamer is as shown in SEQ ID NO:1, and the nucleotide sequence of the DNA is as shown in SEQ ID NO:2.

[0018] The second aspect of the present invention provides a probe for detecting mycotoxins, which is prepared by using the preparation method described in the first aspect above.

[0019] The third aspect of the present invention provides an application of the probe for detecting mycotoxins described in the second aspect in the detection of aflatoxin B1.

[0020] The fourth aspect of the present invention provides a method for detecting aflatoxin B1, comprising the following steps:

[0021] S1 Mix the test substance with a solvent for extraction, and obtain a test solution after solid-liquid separation III;

[0022] S2 Mix the test solution with a probe and let it stand, then perform solid-liquid separation IV, and mix the solid obtained by solid-liquid separation with a fluorescent substrate, metal ions, and water for reaction to obtain a reaction solution, and measure the fluorescence intensity of the reaction solution;

[0023] The probe is the probe for detecting mycotoxin described in the second aspect above.

[0024] Preferably, the method further includes: measuring the fluorescence intensity of a standard solution and plotting a standard curve, calculating the concentration of aflatoxin B1 according to the standard curve, and calculating the content of aflatoxin B1 in the test substance according to the concentration of aflatoxin B1, the volume of the reaction solution, and the mass of the test substance.

[0025] Preferably, in step S1, the solvent is an aqueous solution of methanol.

[0026] More preferably, in step S2, the conditions for standing include: temperature is 20 - 40 °C, and time is 1 - 2 h.

[0027] Preferably, the solid-liquid separation IV is centrifugal separation, and the conditions for centrifugal separation include: rotation speed 8000 - 10000 rpm, and time is 6 - 8 min.

[0028] Preferably, the metal ions are selected from at least one of divalent cadmium ions, divalent lead ions, and trivalent aluminum ions.

[0029] Preferably, in step S1, the preparation method of the fluorescent substrate is: mix HAuCl4, glutathione (GSH), and water to obtain a mixed solution, adjust the pH of the mixed solution to 7 - 9, and obtain the fluorescent substrate after stabilization.

[0030] More preferably, the stabilization time is 1 - 2 h.

[0031] Through the above technical solution, the preparation method provided by the present invention uses an EDTA salt as a carrier for the nucleic acid aptamer, and at the same time uses silica as a carrier for the DNA complementary to the nucleic acid aptamer, and reacts the two, so that the prepared probe not only has a wide applicable temperature range and stability, but also has high sensitivity, effectively improving the detection limit and detection rate of the detected substance, providing a new way for the immunodetection analysis method to be used for the detection of food hazards. At the same time, the probe can be extended to the rapid and highly sensitive detection of other trace and trace hazards in food, providing a scientific basis for preventing food safety accidents and ensuring food health. Description of the Drawings

[0032] Figure 1 is the schematic diagram of the detection method shown in the present invention;

[0033] Figure 2 is the fluorescence spectrogram of the standard sample of AFB1 detected in Example 1;

[0034] Figure 3 is the standard curve of the standard sample of AFB1 detected in Example 1;

[0035] Figure 4 is the specificity study diagram in Example 5;

[0036] Figure 5 is the effect of different concentrations of Cd 2+ on the fluorescence intensity of Au NCs at 555 nm;

[0037] Figure 6 is the fluorescence intensity of Au NCs generated by regulating different concentrations of EDTA·2Na at 555 nm in Example 7. Detailed Embodiments

[0038] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0039] As described above, the first aspect of the present invention provides a method for preparing a probe for detecting mycotoxins, including the following steps:

[0040] (1) Mix a coupling agent I, an ETDA salt and water, and carry out an activation reaction I to obtain an activation reaction solution I. Mix the activation reaction solution I and the nucleic acid aptamer corresponding to the detected substance to carry out a contact reaction I to obtain an aqueous solution containing an EDTA salt@Apt probe;

[0041] Couple coupling agent II, silica, and water to carry out activation reaction II to obtain an activation reaction solution II. Mix the activation reaction solution II with DNA complementary to the aptamer to carry out contact reaction II, and obtain a SiO2@DNA probe after solid-liquid separation I.

[0042] (2) Mix the solution containing the EDTA salt@Apt probe, the SiO2@DNA probe, and water to carry out contact reaction III, and obtain the probe after solid-liquid separation II.

[0043] Specifically, solid-liquid separation I and solid-liquid separation II can each independently be selected from solid-liquid separation methods such as filtration, suction filtration, centrifugation, etc. Preferably, both solid-liquid separation I and solid-liquid separation II are centrifugal separation, and the conditions for centrifugal separation include: a rotation speed of 8000 - 10000 rpm and a time of 6 - 10 min.

[0044] The aptamer and the DNA can be obtained through commercial purchase or can be synthesized artificially.

[0045] In step (2), the SiO2@DNA probe can first be dissolved in water to obtain a solution containing the SiO2@DNA probe, and then the obtained solution containing the SiO2@DNA probe can be mixed and reacted with the solution containing the EDTA salt@Apt probe. The SiO2@DNA probe can also first be dissolved in the solution containing the EDTA salt@Apt probe, and then water can be added for mixing and reaction. Or first mix the solution containing the EDTA salt@Apt probe and water, and then add the SiO2@DNA probe for mixing and reaction. Preferably, first dissolve the SiO2@DNA probe in water to obtain a solution containing the SiO2@DNA probe, and then mix and react the solution containing the SiO2@DNA probe with the solution containing the EDTA salt@Apt probe. First dispersing the SiO2@DNA probe in water and then contacting it with the EDTA salt@Apt probe in the solution can make the reaction more sufficient.

[0046] The inventors of the present invention found during the research process that using an EDTA salt as a carrier for the aptamer and simultaneously using silica as a carrier for the DNA that is complementary to the aptamer, reacting the above two substances can make the binding between the EDTA salt and the aptamer more stable, thereby enabling the probe prepared by the preparation method to have a wider applicable temperature range and stability, and also having higher sensitivity, effectively improving the detection limit and detection rate of the detected substance. It provides a new probe for the detection of mycotoxins.

[0047] In order to further improve the stability of the prepared probe, preferably, in step (1), the coupling agent I and the coupling agent II are N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, respectively. To further improve the stability of the prepared probe, preferably, in the coupling agent I and the coupling agent II, the molar ratio of N-hydroxysuccinimide to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is independently 1:0.5 - 2, preferably 1:0.5 - 1.2. Specifically, in the coupling agent I, the molar ratio of N-hydroxysuccinimide to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:0.5 - 2; in the coupling agent II, the molar ratio of N-hydroxysuccinimide to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:0.5 - 2.

[0048] The EDTA salt can be any water-soluble EDTA salt. Specifically, it can be potassium EDTA, sodium EDTA, calcium EDTA, magnesium EGTA, etc. In order to further improve the sensitivity of the prepared probe, preferably, the EDTA salt is potassium EDTA and / or sodium EDTA. More preferably, the EDTA salt is disodium EDTA.

[0049] Preferably, the molar ratio of the EDTA salt to the aptamer is 40 - 80:1. The molar ratio of the DNA to the silica is 1:1000 - 5000. The addition amount of the coupling agent I is 0.1 - 0.5 g relative to 1 μmol of the aptamer; the addition amount of the coupling agent II is 0.01 - 0.05 g relative to 1 nmol of the DNA. Under the above ratios, there is a better coupling effect between the EDTA salt and the aptamer corresponding to the detection substance, and between the silica and the DNA complementary to the aptamer, thereby further improving the stability of the prepared probe.

[0050] Preferably, in step (1), the time of the activation reaction I is 10 - 25 min, and the time of the contact reaction I is 1 - 3 h; the time of the activation reaction II is 10 - 25 min, and the time of the contact reaction II is 1 - 3 h. Under the above conditions, there is a better coupling effect between the EDTA salt and the aptamer corresponding to the detection substance, and between the silica and the DNA complementary to the aptamer, thereby further improving the stability of the prepared probe. To further improve the stability of the probe, preferably, in step (2), the time of the contact reaction III is 20 - 40 min.

[0051] In order to further improve the sensitivity and stability of the prepared probe, preferably, the molar ratio of the EDTA salt@Apt probe to the SiO2@DNA probe is 1:4 - 16, specifically it can be 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16 or any value between these values.

[0052] The analyte can be ochratoxin A, aflatoxin B1 or zearalenone. Preferably, the analyte is aflatoxin B1. The above probe has higher sensitivity and detection range for the detection of aflatoxin B1.

[0053] In order to further improve the detection sensitivity of the probe, preferably, the nucleotide sequence of the nucleic acid aptamer is as shown in SEQ ID NO:1; the DNA is a 5'-amino modified nucleotide, and its nucleotide sequence is as shown in SEQ ID NO:2.

[0054] Specifically, the structure of the nucleic acid aptamer is:

[0055] 5’-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-3’(SEQ ID NO:1);

[0056] The structure of the DNA is: 5’-NH2-TTTTTTGGGCCTAGC-3’.

[0057] In a second aspect, the present invention provides a probe for detecting mycotoxins, which is prepared by the preparation method described in the above first aspect. This probe has a wide applicable temperature range and stability, and has high sensitivity.

[0058] According to the present invention, the preparation method of the probe has been described in detail above and will not be elaborated here.

[0059] In a third aspect, the present invention provides an application of the probe for detecting mycotoxins described in the second aspect in the detection of aflatoxin B1. Applying the above probe to the detection of aflatoxin B1 has a wider applicable temperature range and stability, and has higher sensitivity.

[0060] In a fourth aspect, the present invention provides a method for detecting aflatoxin B1, including the following steps:

[0061] S1 Mix the sample to be detected with a solvent for extraction, and obtain the sample solution to be detected after solid-liquid separation II;

[0062] S2 mixes the liquid to be detected and the probe, then allows it to stand, followed by solid-liquid separation IV. The solid obtained from the solid-liquid separation is mixed and reacted with a fluorescent substrate, metal ions, and water to obtain a reaction solution, and the fluorescence intensity of the reaction solution is measured.

[0063] The probe is the probe for detecting mycotoxin described in the second aspect above. Its detection principle is shown in Table 1.

[0064] In step S1, the extraction method can be any one of solvent extraction methods, such as solvent immersion method or Soxhlet extraction method. The former directly immerses the object to be detected in the solvent, so that aflatoxin B1 in the object to be detected gradually dissolves in the solvent and is "soaked" out. The latter uses the principle of solvent heating reflux and siphon to extract aflatoxin B1 in the object to be detected by the solvent. Preferably, the extraction method is the immersion extraction method. Preferably, the immersion extraction time is 10 - 30 min. To further improve the extraction effect, preferably, before mixing the object to be detected and the organic solvent, the object to be detected is crushed into powder. Further preferably, during the extraction process, the mixture of the object to be detected and the organic solvent is shaken or stirred to make the contact between the object to be detected and the organic solvent more sufficient, thereby effectively extracting aflatoxin B1 in the object to be detected.

[0065] The above detection method has high sensitivity for the detection of aflatoxin B1, and has high environmental stability, effectively improving the detection limit and detection rate of the detected substances, providing a new way for the immunoassay method to be used for the detection of food hazards. At the same time, this method can be extended to the rapid and highly sensitive detection of other trace and micro hazards in food, providing a scientific basis for preventing food safety accidents and ensuring food health.

[0066] According to the present invention, preferably, the method further includes: measuring the fluorescence intensity of the standard solution and plotting a standard curve, calculating the concentration of aflatoxin B1 according to the standard curve, and calculating the content of aflatoxin B1 in the object to be detected according to the concentration of aflatoxin B1, the volume of the reaction solution, and the mass of the object to be detected.

[0067] In step S1, the solvent can be an aqueous solution of C1 - C6 alcohols, chloroform, acetonitrile, or water. C1 - C6 alcohols can specifically be methanol, ethanol, ethylene glycol, propanol, 1,3 - propanediol, 1,2 - propanediol, glycerol, 1 - n - butanol, 2 - n - butanol, 1 - isobutanol, 2 - isobutanol, n - pentanol, etc. To further improve the extraction effect of aflatoxin B1 in the object to be detected, preferably, the solvent is an aqueous solution of C1 - C4 monohydric alcohols. Further preferably, it is an aqueous solution of methanol. Preferably, the content of methanol in the aqueous solution of methanol is 50 - 75 vol%.

[0068] The addition amounts of the analyte to be detected and the solvent can be determined according to actual situations. Preferably, relative to 1 g of the analyte to be detected, the addition amount of the solvent is 5 - 8 mL.

[0069] Preferably, in step S2, the conditions for standing still include: the temperature is 20 - 40°C, and the time is 1 - 2 h. Under these standing still conditions, aflatoxin B1 and the probe have a better reaction effect, thereby improving the detection sensitivity and accuracy of aflatoxin B1.

[0070] Solid-liquid separation III and solid-liquid separation IV can each independently be selected from solid-liquid separation methods such as filtration, suction filtration, centrifugation, etc. Preferably, both solid-liquid separation III and solid-liquid separation IV are centrifugal separation. The conditions for solid-liquid separation III include: the rotation speed is 8000 - 10000 rpm, and the time is 6 - 8 min; the conditions for solid-liquid separation IV include: the rotation speed is 3500 - 5000 rpm, and the time is 4 - 8 min.

[0071] The metal ion can be an ion that has a fluorescence effect when reacting with the substrate in the prior art. Specifically, it can be provided through its salt solution. Preferably, the metal ion is selected from at least one of divalent cadmium ions, divalent lead ions, and trivalent aluminum ions. Selecting the above ions to react with the substrate has a better fluorescence effect, thereby further improving the detection sensitivity. Further preferably, the metal ion is divalent cadmium ion. The divalent cadmium ion can be provided by any divalent cadmium salt that is soluble in water, specifically cadmium chloride; the divalent lead ion can be provided by any divalent lead salt that is soluble in water, specifically lead acetate; the trivalent aluminum ion can be provided by any trivalent chloride salt that is soluble in water, specifically aluminum chloride.

[0072] The fluorescent substrate can be any fluorescent substrate prepared by any method disclosed in the prior art. In order to further improve the detection sensitivity and stability, preferably, in step S1, the preparation method of the fluorescent substrate is: mixing HAuCl4, GSH, and water to obtain a mixed solution, adjusting the pH of the mixed solution to 7 - 9, and obtaining the fluorescent substrate after stabilization. Considering further improving the detection sensitivity and stability, preferably, the stabilization time is 1 - 2 h. Preferably, the molar ratio of HAuCl4 to GSH is 1:3 - 5.

[0073] Preferably, relative to 1.5 mg of the probe, the addition amount of the fluorescent substrate is 1 - 4 μmol, and the addition amount of the metal ion is 2 - 8 μmol.

[0074] According to a particularly preferred embodiment of the present invention, a method for detecting aflatoxin B1 is provided, as Figure 1 shown, including the following steps:

[0075] Preparation of the probe:

[0076] (1) Mix N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, disodium ETDA and water, and carry out an activation reaction for 10 - 25 min to obtain activation reaction solution I. Mix the activation reaction solution I with the nucleic acid aptamer corresponding to the detection substance, and incubate at room temperature for 1 - 3 h to obtain a solution containing the EDTA salt@Apt probe; the molar ratio of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, disodium ETDA and the nucleic acid aptamer is 300 - 1500:300 - 2000:40 - 80:1, and the nucleotide sequence of the nucleic acid aptamer is as shown in SEQ ID NO:1;

[0077] Mix N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, silica and water, and carry out an activation reaction for 10 - 25 min to obtain activation reaction solution II. Mix the activation reaction solution II with the DNA complementary to the nucleic acid aptamer, and incubate at room temperature for 1 - 3 h. Then, centrifuge at a rotation speed of 8000 - 10000 rpm for 6 - 8 min, and the obtained solid is the SiO2@DNA probe; the molar ratio of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, silica and DNA is 30000 - 150000:30000 - 200000:1000 - 5000:1, and the DNA is 5'-amino-modified nucleotides, and its nucleotide sequence is as shown in SEQ ID NO:2;

[0078] (2) Mix the solution containing the EDTA salt@Apt probe, the SiO2@DNA probe and water, and react at 30 - 40 °C for 20 - 40 min. Then, centrifuge at a rotation speed of 8000 - 10000 rpm for 6 - 8 min, and the obtained solid is the probe; the molar ratio of the EDTA salt@Apt probe to the SiO2@DNA probe is 1:4 - 16.

[0079] Preparation of the fluorescent substrate:

[0080] Mix HAuCl4, GSH and water to obtain a mixed solution, adjust the pH of the mixed solution to 7 - 9, and stabilize for 1 - 2 h to obtain the fluorescent substrate, and the molar ratio of HAuCl4 to GSH is 1:3 - 5.

[0081] S1 Mix the substance to be detected and an aqueous methanol solution for extraction for 10 - 30 min, and centrifuge at a rotation speed of 8000 - 10000 rpm for 6 - 8 min. The obtained liquid is the liquid to be detected;

[0082] S2 mixes the liquid to be detected and the probe, then stands them at a temperature of 20 - 40°C for 1 - 2 h, centrifuges them at a rotational speed of 3500 - 5000 rpm for 4 - 8 min, and mixes the obtained solid after centrifugation with a fluorescent substrate, divalent cadmium ions, and water to obtain a reaction solution, and measures the fluorescence intensity of the reaction solution;

[0083] Measure the fluorescence intensity of the standard solution and plot a standard curve, calculate the concentration of aflatoxin B1 according to the standard curve, and calculate the content of aflatoxin B1 in the substance to be detected based on the concentration of aflatoxin B1, the volume of the reaction solution, and the mass of the substance to be detected;

[0084] Relative to 1.5 mg of the probe, the addition amount of the fluorescent substrate is 1 - 4 μmol, and the addition amount of the metal ions is 2 - 8 μmol.

[0085] The present invention will be described in detail below through examples. In the following examples, 5’-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-3’ is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., 5’-NH2-TTTTTTGGGCCTAGC-3’ is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., 5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-C6H 12 -NH2-3’ is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., 5’-NH2-C6H 12 -TGTCCGATGCT-3’ is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., N-hydroxysuccinimide is purchased from Beijing Solarbio Science & Technology Co., Ltd., and aflatoxin B1 and ochratoxin A are obtained through commercial purchase.

[0086] The fluorescence spectrophotometer is purchased from Hitachi Ltd., and the instrument model is F-7000; the kit is purchased from Beijing Huaan Maike Biotechnology Co., Ltd., and the usage process is carried out according to the instructions.

[0087] Preparation Example A1

[0088] (1) Mix 1.5 mmol of N-hydroxysuccinimide, 0.9 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 60 μmol of disodium EDTA and 48 mL of water, and carry out an activation reaction for 15 min to obtain activation reaction solution I. Mix the activation reaction solution I with 1 μmol of aptamer and incubate at room temperature for 1 h to obtain a solution containing the EDTA salt@Apt probe, wherein the concentration of the EDTA salt@Apt probe is 0.1 mM; the nucleotide sequence of the aptamer is as shown in SEQ ID NO:1;

[0089] Mix 1.5 mmol of N-hydroxysuccinimide, 0.9 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 25 μmol of silica and 17 mL of water, and carry out an activation reaction for 15 min to obtain activation reaction solution II. Mix the activation reaction solution II with 10 nmol of DNA complementary to the aptamer and incubate at room temperature for 1 h, then centrifuge at 10,000 rpm for 8 min, and wash 3 times with PBS buffer (0.01 M, pH 7.2 - 7.4). The obtained solid is the SiO2@DNA probe; the DNA is 5'-amino-modified nucleotides, and its nucleotide sequence is as shown in SEQ ID NO:2;

[0090] (2) Mix 4 μL of 1 μM aqueous solution of the EDTA salt@Apt probe and 16 μL of 10 μM aqueous solution of the SiO2@DNA probe, and react at 37 °C for 30 min, then centrifuge at 10,000 rpm for 8 min, and wash 3 times with PBS buffer (0.01 M, pH 7.2 - 7.4). The obtained solid is the probe, and store it at 4 °C for standby.

[0091] Preparation Example A2

[0092] (1) Mix 0.33 mmol of N-hydroxysuccinimide, 0.32 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 40 μmol of disodium EDTA and 32 mL of water, and carry out an activation reaction for 10 min to obtain activation reaction solution I. Mix the activation reaction solution I with 1 μmol of aptamer and incubate at room temperature for 2 h to obtain a solution containing the EDTA salt@Apt probe, wherein the concentration of the EDTA salt@Apt probe is 0.1 mM; the nucleotide sequence of the aptamer is as shown in SEQ ID NO:1;

[0093] Mix 0.33 mmol of N-hydroxysuccinimide, 0.32 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 10 μmol of silica, and 6 mL of water, and carry out an activation reaction for 10 min to obtain activation reaction solution II. Mix the activation reaction solution II with 10 nmol of DNA complementary to the aptamer, incubate at room temperature for 2 h, then centrifuge at 8000 rpm for 7 min, and wash 3 times with PBS buffer (0.01 M, pH 7.2 - 7.4). The obtained solid is the SiO2@DNA probe; the DNA is a 5'-amino-modified nucleotide, and its nucleotide sequence is as shown in SEQ ID NO:2;

[0094] (2) Mix 5 mL of 1 μM EDTA salt@Apt probe and 5 mL of 4 μM SiO2@DNA probe, react at 30 °C for 40 min, then centrifuge at 8000 rpm for 7 min, and wash 3 times with PBS buffer (0.01 M, pH 7.2 - 7.4). The obtained solid is the probe, and store it at 4 °C for later use.

[0095] Preparation Example A3

[0096] (1) Mix 1 mmol of N-hydroxysuccinimide, 2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 80 μmol of disodium ETDA, and 96 mL of water, and carry out an activation reaction for 25 min to obtain activation reaction solution I. Mix the activation reaction solution I with 1 μmol of aptamer, incubate at room temperature for 3 h to obtain a solution containing EDTA salt@Apt probe, wherein the concentration of the EDTA salt@Apt probe is 0.1 mM; the nucleotide sequence of the aptamer is as shown in SEQ ID NO:1;

[0097] Mix 1 mmol of N-hydroxysuccinimide, 2 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 50 μmol of silica, and 10 mL of water, and carry out an activation reaction for 25 min to obtain activation reaction solution II. Mix the activation reaction solution II with 10 nmol of DNA complementary to the aptamer, incubate at room temperature for 3 h, then centrifuge at 9000 rpm for 6 min, and wash 3 times with PBS buffer (0.01 M, pH 7.2 - 7.4). The obtained solid is the SiO2@DNA probe; the DNA is a 5'-amino-modified nucleotide, and its nucleotide sequence is as shown in SEQ ID NO:2;

[0098] (2) Mix 5 mL of 1 μM EDTA salt @ Apt probe and 5 mL of 16 μM SiO2 @ DNA probe, react at 40 °C for 20 min, then centrifuge at 9000 rpm for 6 min, wash 3 times with PBS buffer (0.01 M, pH 7.2 - 7.4). The obtained solid is the probe, and store it at 4 °C for standby.

[0099] Preparation Example B1

[0100] Mix 0.5 mL of 20 mM HAuCl4, 0.2 mL of 100 mM GSH and 4.2 mL of water to obtain a mixed solution, adjust the pH of the mixed solution to 8, and obtain an aqueous solution containing a fluorescent substrate after stabilizing for 1.5 h.

[0101] Preparation Example B2

[0102] Mix 0.5 mL of 20 mM HAuCl4, 0.2 mL of 50 mM GSH and 4.2 mL of water to obtain a mixed solution, adjust the pH of the mixed solution to 7, and obtain an aqueous solution containing a fluorescent substrate after stabilizing for 2 h.

[0103] Preparation Example B3

[0104] Mix 0.5 mL of 10 mM HAuCl4, 0.2 mL of 100 mM GSH and 4.2 mL of water to obtain a mixed solution, adjust the pH of the mixed solution to 6, and obtain an aqueous solution containing a fluorescent substrate after stabilizing for 1 h.

[0105] Example 1

[0106] Prepare an aqueous probe solution with a concentration of 1.5 mg / mL from the probe prepared in Preparation A1;

[0107] Add 200 μL of the aqueous probe solution and 200 μL of aflatoxin B1 solutions with concentrations of 0, 10 -16 , 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 g / mL to a centrifuge tube, incubate at 30 °C for 1.5 h, then centrifuge at 9000 rmp for 7 min. After discarding the supernatant, dissolve the precipitate in 0.05 mL of Cd 2+ solution (0.8 mM), add 0.25 mL of the fluorescent substrate prepared in Preparation Example B1, react at room temperature for 15 minutes, and then measure the fluorescence intensity at 555 nm with a fluorescence spectrophotometer. Fit a standard curve according to the detection results, see Figure 2and Figure 3 The detection limit and detection range are shown in Table 1.

[0108] Example 2

[0109] The probe prepared in Preparation A2 was formulated into an aqueous probe solution with a concentration of 1.5 mg / mL;

[0110] Add 200 μL of the aqueous probe solution and 200 μL of aflatoxin B1 solutions with concentrations of 0, 10 -16 , 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 g / mL into a centrifuge tube, incubate at 20 °C for 2 h, then centrifuge at 8000 rmp for 8 min. After discarding the supernatant, dissolve the precipitate in 0.05 mL of Cd 2+ solution (0.8 mM), add 0.25 mL of the fluorescent substrate prepared in Preparation Example B2, and measure the fluorescence intensity at 350 nm with a fluorescence spectrophotometer after reacting at room temperature for 10 minutes.

[0111] Example 3

[0112] The probe prepared in Preparation A3 was formulated into an aqueous probe solution with a concentration of 1.5 mg / mL;

[0113] Add 200 μL of the aqueous probe solution and 200 μL of aflatoxin B1 solutions with concentrations of 0, 10 -16 , 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 g / mL into a centrifuge tube, incubate at 40 °C for 1 h, then centrifuge at 10000 rmp for 6 min. After discarding the supernatant, dissolve the precipitate in 0.05 mL of Cd 2+ solution (0.8 mM), add 0.25 mL of the fluorescent substrate prepared in Preparation Example B3, and measure the fluorescence intensity at 555 nm with a fluorescence spectrophotometer after reacting at room temperature for 10 minutes.

[0114] Example 4

[0115] S1 Fit the standard curve according to the method described in Example 1.

[0116] S2 Mix 1 g of expired sesame powder with 25 mL of 60% methanol aqueous solution and extract for 20 min. The liquid obtained by centrifuging for 7 min at a rotational speed of 9000 rpm is the test solution to be detected;

[0117] S3 Mix 200 μL of the test solution to be detected with 200 μL of the probe aqueous solution in Example 1, let it stand at 30 °C for 1.5 h, centrifuge at a rotational speed of 4000 rpm for 6 min, and dissolve the solid obtained by centrifugation in 50 μL of Cd 2+ solution (0.8 mM). Add 0.25 mL of the fluorescent substrate prepared in Preparation Example B1, and after reacting at room temperature for 15 minutes, measure the fluorescence intensity at 555 nm with a fluorescence spectrophotometer;

[0118] S4 Calculate the concentration of aflatoxin B1 according to the standard curve, and calculate the content of aflatoxin B1 in the sesame powder according to the concentration of aflatoxin B1, the volume of the reaction solution, and the mass of the substance to be detected. The values are shown in Table 2.

[0119] Example 5

[0120] Prepare a probe aqueous solution with a concentration of 1.5 mg / mL from the probe prepared in Preparation A1;

[0121] Mix 200 μL of the probe aqueous solution with 200 μL of 10 -9 μg / mL of fumonisin (FB1), ochratoxin A (OTA), deoxynivalenol (DON), aflatoxin M1 (AFM1), T-2 toxin (T-2), aflatoxin B2 (AFB2), aflatoxin B1 (AFB1), and mixed toxins into 8 centrifuge tubes respectively. Incubate at 37 °C for 1 h and then centrifuge at 10000 rpm for 8 minutes. After discarding the supernatant, dissolve the precipitate in 50 μL of 0.8 mM Cd 2+ solution. Then add 0.2 mL of the fluorescent substrate prepared in Preparation Example B1, and after reacting at room temperature for 10 minutes, measure the fluorescence intensity at an incident wavelength of 330 nm and an emission wavelength of 555 nm with a fluorescence spectrophotometer, and analyze the detection results.

[0122] According to the difference between the fluorescence intensity of detecting different mycotoxins and the blank value, draw a bar chart of mycotoxins and fluorescence intensity. The detection results are as Figure 4 shown. The fluorescence intensity of aflatoxin B1 is basically the same as that of the mixed toxins, and is much greater than the blank value. The fluorescence intensities of the remaining toxins approach the blank value, indicating that this detection method has good selectivity.

[0123] Example 6

[0124] To Cd 2+The fluorescent substrate prepared in Preparation Example B3 was added to the solution so that the final concentrations of Cd 2+ were 0, 0.28, 0.44, 0.52, 0.60, 0.68, 0.8, 1, 1.2, 1.4 mM respectively. After reacting at room temperature for 10 minutes, the fluorescence intensity at an emission wavelength of 555 nm when the incident wavelength was 330 nm was measured using a fluorescence spectrophotometer.

[0125] The detection results were as Figure 5 shown. When the concentration of Cd 2+ was 0 - 0.8 mM, the fluorescence intensity increased with the increase in the concentration of Cd 2+ . When the concentration of Cd 2+ was 0.8 mM, the aggregation-induced emission (AIE) effect was the strongest and the fluorescence intensity reached the maximum value.

[0126] Example 7

[0127] 20 μL of disodium EDTA with concentrations of 0, 0.00025, 0.001, 0.02, 0.078, 0.3, 1.25 mM was added to a centrifuge tube, 50 μL of 4 mM Cd 2+ solution was added, and then 200 μL of the fluorescent substrate in Preparation Example B1 was added. After mixing evenly, it was incubated for 10 min, and the fluorescence intensity at an emission wavelength of 555 nm when the incident wavelength was 330 nm was measured using a fluorescence spectrophotometer.

[0128] The detection results were as Figure 6 shown. As the concentration of disodium EDTA increased, the fluorescence intensity of the solution decreased, indicating that disodium EDTA had a chelating effect on Cd 2+ .

[0129] Comparative Example 1

[0130] An expired sesame powder used in Example 4 was detected using a commercial kit for aflatoxin B1, and the content of aflatoxin B1 was shown in Table 2.

[0131] Comparative Example 2

[0132] Graphene quantum dots were synthesized by a hydrothermal method, and 10 mL of graphene quantum dot solution (0.1 mg / mL), 22 mg of N-hydroxysuccinimide, and 19 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were mixed. The pH of the solution was adjusted to 5. After reacting for 30 min, it was divided into two beakers, both 5 mL. 1 μmol of aptamer and 10 nmol of DNA complementary to the aptamer were added respectively (the nucleotide sequence of the aptamer was 5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-C6H 12-NH2-3’, the DNA is a 5’-amino modified nucleotide, and its nucleotide sequence is 5’-NH2-C6H 12 -TGTCCGATGCT-3’), adjust the pH of the solution to 7.4, and react for 2 h;

[0133] Add ochratoxin A to the above solution to make its concentration 0, 10 -16 、10 -15 、10 -14 、10 -13 、10 -12 、10 -11 、10 -10 、10 -9 、10 -8 g / mL, measure the fluorescence intensity at 555 nm with a fluorescence spectrophotometer, fit a standard curve according to the detection results, and the detection limit and detection range are shown in Table 1.

[0134] Table 1

[0135] Detection limit pg / mL Detection range ng / mL Example 1 <![CDATA[2×10 -4 > <![CDATA[10 -6 -10 -1 > Comparative Example 2 13 0-1

[0136] Table 2

[0137] Content of aflatoxin B1 in expired sesame powder (μg / kg) Example 4 47.5 Comparative Example 1 40.53

[0138] It can be seen from the results in Table 1 that the method adopted in the present invention has a lower detection limit and a larger detection range compared with the prior art, indicating that the method provided in the present invention has high sensitivity.

[0139] It can be seen from Table 2 that the amount of aflatoxin B1 detected in the actual sample by the method adopted in the present invention is basically the same as that of the kit used in the market, indicating that the probe in the present invention has good accuracy for detecting aflatoxin B1.

[0140] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A method for preparing a probe for detecting mycotoxin, characterized in that, It includes the following steps: (1) Mix coupling agent I, ETDA salt and water to carry out activation reaction I to obtain activation reaction solution I. Mix the activation reaction solution I with the nucleic acid aptamer corresponding to the detection substance to carry out contact reaction I to obtain a solution containing EDTA salt@Apt probe; Mix coupling agent II, silicon dioxide and water to carry out activation reaction II to obtain activation reaction solution II. Mix the activation reaction solution II with the DNA complementary to the nucleic acid aptamer to carry out contact reaction II. After solid-liquid separation I, obtain SiO2@DNA probe; (2) Mix the solution containing EDTA salt@Apt probe, the SiO2@DNA probe and water, then carry out contact reaction III. After solid-liquid separation II, obtain the probe.

2. The preparation method according to claim 1, wherein, In step (1), the coupling agent I and the coupling agent II are N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride respectively.

3. The preparation method according to claim 2, characterized in that, Among the coupling agent I and the coupling agent II, the molar ratio of the N-hydroxysuccinimide and the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is independently 1:0.5-2; The EDTA salt is potassium EDTA and / or sodium EDTA.

4. The preparation method according to claim 1, characterized in that, In step (1), the time of activation reaction I is 10-25 min, and the time of contact reaction I is 1-3 h; The time of activation reaction II is 10-25 min, and the time of contact reaction II is 1-3 h; In step (2), the time of contact reaction III is 20-40 min; The molar ratio of the EDTA salt@Apt probe and the SiO2@DNA probe is 1:4-16.

5. The preparation method according to any one of claims 1-4, characterized in that, The detection substance is aflatoxin B1; The nucleotide sequence of the nucleic acid aptamer is as shown in SEQ ID NO:1; the DNA is 5'-amino-modified nucleotide, and its nucleotide sequence is as shown in SEQ ID NO:

2.

6. A probe for detecting mycotoxin, characterized in that, The probe is prepared by the preparation method described in any one of claims 1-5.

7. Application of the probe for detecting mycotoxin described in claim 6 in detecting aflatoxin B1.

8. A detection method for aflatoxin B1, characterized in that, It includes the following steps: S1 Mix the substance to be detected and a solvent for extraction. After solid-liquid separation III, obtain the liquid to be detected; S2 Mix the liquid to be detected and the probe, then let it stand. Then carry out solid-liquid separation IV, and mix the solid obtained by solid-liquid separation with a fluorescent substrate, metal ions and water for reaction to obtain a reaction solution, and measure the fluorescence intensity of the reaction solution; The probe is the probe for detecting mycotoxin described in claim 6.

9. The detection method according to claim 8, wherein The method further includes: measuring the fluorescence intensity of a standard solution and plotting a standard curve, calculating the concentration of aflatoxin B1 according to the standard curve, and calculating the content of aflatoxin B1 in the substance to be detected according to the concentration of the aflatoxin B1, the volume of the reaction solution and the mass of the substance to be detected.

10. The detection method according to claim 8, wherein In step S1, the solvent is an aqueous solution of methanol; In step S2, the standing conditions include: temperature is 20-40 °C, and time is 1-2 h; The metal ions are selected from at least one of divalent cadmium ions, divalent lead ions and trivalent aluminum ions.

11. The detection method according to any one of claims 8-10, characterized in that, In step S1, the preparation method of the fluorescent substrate is as follows: mixing HAuCl4, glutathione and water to obtain a mixed solution, adjusting the pH of the mixed solution to 7-9, and obtaining the fluorescent substrate after stabilization.

12. The detection method according to claim 11, wherein The stabilization time is 1-2 h.

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