Method for detecting mycotoxins based on the crisper-cas12a system

Abstract

The present application relates to the field of electrochemical analysis and detection, and discloses a method for detecting mycotoxin based on CRISPR-Cas12a system, comprising: (1) reacting single-stranded DNA with recognition DNA of mycotoxin to obtain single-stranded-recognition DNA complex, wherein the single-stranded DNA is complementary to the recognition DNA; (2) mixing the single-stranded-recognition DNA complex and the sample to be tested which may contain mycotoxin to obtain reaction mixture I after reaction II, and mixing the reaction mixture I with auxiliary DNA containing activated DNA fragments to obtain reaction mixture II after reaction III, wherein the auxiliary DNA is complementary to the single-stranded DNA; (3) mixing the reaction mixture II with CRISPR-Cas12a system solution to obtain reaction solution after activation; (4) adding the reaction solution dropwise to an electrochemical biosensor to perform reaction V, and detecting the electrochemical signal of the electrochemical biosensor after reaction V is completed. The method has high sensitivity and high selectivity, and is simple and rapid to operate.

Description

Technical Field

[0001] This invention relates to the field of electrochemical analysis and detection, specifically to a method for detecting fungal toxins based on the CRISPR-Cas12a system. Background Technology

[0002] Ochratoxin A (OTA) is one of the most common and highly toxic mycotoxins in food, capable of causing liver, kidney, neurological, and immune damage, and is also a carcinogen. Human exposure to OTA primarily occurs through the consumption of improperly stored food, especially contaminated grains. Due to its high physical and chemical stability, OTA is almost impossible to completely degrade under normal cooking conditions (such as high temperatures) when contaminated food is ingested. Therefore, once ingested, OTA can accumulate in the body over a long period, potentially causing irreversible toxic effects on life and health. Because of the widespread presence of OTA in various food matrices, the European Commission has regulated permissible limits for OTA in various foods, such as grains (5 μg / kg), wine (2 μg / kg), and grape juice (2 μg / kg). In this context, the detection of trace amounts of OTA in food is particularly important. Currently, many methods, including high-performance liquid chromatography (HPLC), mass spectrometry (MS), fluorescence methods, surface-enhanced Raman scattering (SMR), and colorimetry, have been used for OTA detection. While these methods offer relatively high accuracy, they often require complex operations and expensive instruments, and suffer from poor applicability in complex food matrices. Considering the high toxicity of OTA and its presence in complex food matrices, OTA detection necessitates higher specificity and sensitivity. Therefore, there is an urgent need to establish a robust, accurate, widely applicable, and ultrasensitive method for the detection of OTA in food.

[0003] In recent years, the discovery and development of clustered regularly spaced short palindromic repeats (CRISPR) and their associated (Cas) systems have not only revolutionized gene editing but also had a profound impact on other fields. Cas12a, belonging to the CRISPR-Cas effector family, possesses the ability to target and cleave double-stranded DNA and single-stranded DNA (ssDNA) via cis-cleavage after recognizing its corresponding RNA (crRNA), and also exhibits trans-cleavage activity on the side chains of any surrounding ssDNA. Utilizing these properties, CRISPR-Cas12a has been used to develop numerous molecular diagnostic strategies, such as the CRISPR-Cas12a-based nucleic acid sensors SHERLOCK and DETECTR. Furthermore, depending on the detection target, CRISPR-Cas12a-based diagnostic methods have expanded from nucleic acids to non-nucleic acid targets, such as enzyme detection, biomarkers, metal ion detection, and small molecule detection. Based on sensor type, CRISPR-Cas12a biosensors mainly include fluorescence sensors, colorimetric sensors, and electrochemical sensors. However, most CRISPR-Cas12a-based biosensors rely on traditional fluorescence quenching probes, which are prone to oxidation, have low sensitivity, and must be protected from light, making them unsuitable for practical applications. Given that CRISPR-Cas technology is not yet fully utilized in food detection, and considering the complexity of food matrices and the high sensitivity required for food-related detection, this technology is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to overcome the problem that the sensitivity of existing biosensors needs to be improved, and to provide a method for detecting fungal toxins. This method has high sensitivity and high selectivity, and is simple and fast to operate.

[0005] To achieve the above objectives, the present invention provides a method for detecting mycotoxins based on the CRISPR-Cas12a system, comprising the following steps:

[0006] (1) Reaction I of single-stranded DNA with the recognition DNA of fungal toxins yields a single-stranded-recognition DNA complex, wherein the single-stranded DNA is complementary to the recognition DNA;

[0007] (2) After mixing the single-stranded recognition DNA complex with a test sample that may contain fungal toxins for reaction II, it is mixed with an auxiliary DNA containing an activating DNA fragment for reaction III to obtain reaction mixture I. The reaction mixture I is mixed with an auxiliary enzyme for reaction IV to obtain reaction mixture II that may contain the activating DNA. The auxiliary DNA is DNA complementary to the single-stranded DNA.

[0008] (3) The reaction mixture II was mixed with a CRISPR-Cas12a system solution for activation to obtain a reaction solution;

[0009] (4) The reaction solution obtained in step (3) is added dropwise to the electrochemical biosensor to carry out reaction V. After the reaction V is completed, the electrochemical signal of the electrochemical biosensor is detected.

[0010] The helper DNA can bind to the single-stranded DNA to form a single-stranded helper DNA complex, and the helper enzyme can digest the single-stranded helper DNA complex to release the activated DNA.

[0011] Preferably, the fungal toxin is a mycotoxin.

[0012] More preferably, the mycotoxin is ochratoxin A, the nucleotide sequence of the single-stranded DNA is shown in SEQ ID No. 1, the nucleotide sequence of the recognition DNA is shown in SEQ ID No. 2, and the nucleotide sequence of the helper DNA is shown in SEQ ID No. 3.

[0013] Preferably, in step (1), the molar ratio of the recognition DNA to the single-stranded DNA is 1:1.25-1.75.

[0014] More preferably, the conditions for reaction I include: a temperature of 90-98°C and a time of 3-8 minutes.

[0015] Preferably, in step (2), the amount of auxiliary DNA added is 1-1.2 mol relative to 1 mol of single-stranded DNA.

[0016] More preferably, in step (2), the conditions for reaction II include: a temperature of 30-45°C and a time of 60-90 min;

[0017] The conditions for reaction III include: a temperature of 30-45°C and a time of 30-60 min.

[0018] Preferably, in step (2), the coenzyme is capable of digesting the single-stranded helper DNA complex to release the activated DNA and the single-stranded DNA, and is preferably an Exo III enzyme.

[0019] Preferably, when the reaction mixture II contains the single-stranded DNA, the method further includes: continuing the reaction of the single-stranded DNA, which may be present, in the reaction mixture II.

[0020] Preferably, in step (3), the CRISPR-Cas12a system solution contains crRNA, Cas12a protein, ribonuclease inhibitor and buffer; wherein the nucleotide sequence of the crRNA is shown in SEQ ID No. 5, and the nucleic acid sequence of the activating DNA is shown in SEQ ID No. 4.

[0021] Preferably, in step (3), the activation conditions include: a temperature of 30-45°C and a time of 20-30 min.

[0022] Preferably, the electrochemical biosensor includes an electrode and a functional layer modified on the electrode, the functional layer containing polyacrylamide hydrochloride, cerium dioxide, and ferrocene-labeled nonspecific single-stranded DNA.

[0023] More preferably, the molar ratio of the polyacrylamide hydrochloride, the cerium dioxide, and the ferrocene-labeled nonspecific single-stranded DNA is 1 × 10⁻⁶. 4 -2×10 4 1.5×10 3 -4×10 3 :1.

[0024] More preferably, the nucleotide sequence of the nonspecific single-stranded DNA is shown in SEQ ID No. 6.

[0025] Preferably, the electrode is a glassy carbon electrode, a gold electrode, or a platinum electrode.

[0026] Preferably, the method further includes: before step (4), detecting the initial electrochemical signal of the electrochemical biosensor and calculating the percentage difference between the electrochemical signal and the initial electrochemical signal.

[0027] The beneficial effects of the present invention through the above technical solution are as follows:

[0028] (1) The method provided by this invention has good detection specificity and high sensitivity, within 1×10 -6 It exhibits good linearity within the range of -5 ng / mL, with a limit of detection of 0.74 fg / mL, and the detection results are accurate and reliable.

[0029] (2) The method provided by the present invention is simple to operate and does not require complex instruments. The operating temperature of Cas12a is 37°C, and it does not require complex temperature variation conditions. Therefore, the requirements for instruments are not high and it is easy to operate.

[0030] (3) The method provided by the present invention is not only applicable to the detection of ochratoxin A in food, but also has great application potential in the detection of other fungal toxins.

[0031] (4) In this invention, after using the ExoIII enzyme, single-stranded auxiliary DNA can release single-stranded DNA under the action of the ExoIII enzyme. This single-stranded DNA can continue to react with auxiliary DNA to form single-stranded auxiliary DNA. The formed single-stranded auxiliary DNA can continue to act with the ExoIII enzyme to realize the recycling of single-stranded DNA, thereby improving the utilization rate of single-stranded DNA. Moreover, it can generate a large amount of activated DNA that binds to crRNA in the CRISPR-Cas12a system, amplify the trans-cutting efficiency of CRISPR-Cas12a, and improve the signal intensity generated by the signal probe in the electrochemical biosensor after cutting. This can effectively increase the detection sensitivity and quantitatively analyze the ochratoxin A in the sample by changing the electrochemical signal response value. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the method for detecting ochratoxin A provided by the present invention;

[0033] Figure 2 The graphs show the CV response results of the modified electrodes in Example 1 and Comparative Example 1, where a corresponds to the PAH & Fc-ssDNA modified electrode (Comparative Example 1) and b corresponds to the PAH-CeO2 & Fc-ssDNA modified electrode (Example 1).

[0034] Figure 3 Figure A shows the analysis and detection results of the feasibility of the electrochemical biosensor in Example 2. Figure B is a polyacrylamide gel electrophoresis diagram of the reaction solution and a polyacrylamide gel electrophoresis diagram of the CRISPR-Cas12a system cleavage activity verification.

[0035] Figure 4 These are peak current variation graphs from Examples 2 to 7, where Figure A corresponds to the concentration of cerium dioxide, Figure B corresponds to the concentration of polyacrylamide hydrochloride, Figure C corresponds to the concentration of Fc-ssDNA, Figure D corresponds to the concentration of Cas12a, Figure E corresponds to the concentration of crRNA, and Figure F corresponds to the cleavage time of the activated CRISPR-Cas12a system on the sensor surface.

[0036] Figure 5 Figure 8 shows the detection results of different concentrations of ochratoxin A in Example 8. Figure A shows the DPV response results of the electrochemical biosensor to different concentrations of ochratoxin A, and Figure B shows the standard curve of the peak current change value of DPV versus the logarithm of the concentration of ochratoxin A.

[0037] Figure 6 This is a graph showing the specific detection results in Example 9. Detailed Implementation

[0038] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0039] This invention provides a method for detecting mycotoxins based on the CRISPR-Cas12a system, comprising the following steps:

[0040] (1) Reaction I of single-stranded DNA with the recognition DNA of fungal toxins yields a single-stranded-recognition DNA complex, wherein the single-stranded DNA is complementary to the recognition DNA;

[0041] (2) After mixing the single-stranded recognition DNA complex with a test sample that may contain fungal toxins for reaction II, it is mixed with an auxiliary DNA containing an activating DNA fragment for reaction III to obtain reaction mixture I. The reaction mixture I is mixed with an auxiliary enzyme for reaction IV to obtain reaction mixture II that may contain the activating DNA. The auxiliary DNA is DNA complementary to the single-stranded DNA.

[0042] (3) The reaction mixture II was mixed with a CRISPR-Cas12a system solution for activation to obtain a reaction solution;

[0043] (4) The reaction solution obtained in step (3) is added dropwise to the electrochemical biosensor to carry out reaction V. After the reaction V is completed, the electrochemical signal of the electrochemical biosensor is detected.

[0044] The helper DNA can bind to the single-stranded DNA to form a single-stranded helper DNA complex, and the helper enzyme can digest the single-stranded helper DNA complex to release the activated DNA.

[0045] Specifically, the single-stranded-recognition DNA complex is a complex formed by single-stranded DNA and recognition DNA. Auxiliary DNA containing an activating DNA fragment and single-stranded DNA can react to form a single-stranded-auxiliary DNA complex. The complementarity mentioned in this invention is not complementarity in the conventional sense, but rather refers to a portion of one DNA segment being complementary to another DNA segment. For example, the single-stranded DNA is complementary to a portion of the recognition DNA, and the single-stranded DNA is complementary to a portion of the auxiliary DNA.

[0046] According to the method provided by this invention, an initial electrochemical signal belonging to an electrochemical biosensor can be detected in the absence of mycotoxins. When mycotoxins are present, they are recognized and bound by the recognition DNA in the single-stranded recognition DNA complex, thereby releasing single-stranded DNA. This released single-stranded DNA reacts with helper DNA containing an activating DNA fragment to form a single-stranded helper DNA complex. Under the action of a helper enzyme, this complex generates activating DNA, which further binds to the CRISPR-Cas12a system to activate the trans-cleavage activity of Cas12a. When the activated CRISPR-Cas12a system is introduced into the electrochemical biosensor, the probes on the sensor are cleaved, resulting in a sharp decrease in the electrochemical signal.

[0047] According to the present invention, an electrochemical biosensor is placed in a detection solution, and its electrochemical signal can be obtained by measuring the change in current peak value with potential using differential pulse voltammetry (DPV). The detection solution can be a Tris-HCl buffer solution with a concentration of 0.05-0.15 mol / L (containing 0.1-0.3 mol / L NaCl) and a pH of approximately 7.4; the detection range of the differential pulse voltammetry can be -0.2V to 0.7V.

[0048] The method of this invention can be used for both qualitative and quantitative detection. For qualitative detection, the signal of a blank sample under the same environment is compared with the detection signal. If a difference exists between the detection signal and the blank sample signal, the sample contains mycotoxins. For quantitative detection, the electrochemical signals of mycotoxins at different concentrations are first detected using the above method, and a standard curve is plotted. Then, the electrochemical signal of the sample to be tested is detected, and the content of mycotoxins in the sample is calculated based on the detected electrochemical signal, the standard curve, the sample solution volume, and the sample itself. Preferably, the method provided by this invention is applied to quantitative detection.

[0049] The detection method employed in this invention exhibits high specificity and provides accurate and reliable results; when used for quantitative detection, its detection range is 1×10⁻⁶. -6 The detection limit is 0.74 fg / mL, which is -5 ng / mL, indicating that the detection method of the present invention has high sensitivity, wide detection range and low detection limit.

[0050] The mycotoxin can be any type of mycotoxin, specifically vomitoxin, zearalenone, fumonisin, T-2 toxin, or aflatoxin B1. Preferably, the mycotoxin is a mycotoxin, and the mycotoxin can be any one of fumonisin, aflatoxin B1, and ochratoxin A.

[0051] More preferably, the mycotoxin is ochratoxin A (OTA), the nucleotide sequence of the single-stranded DNA is shown in SEQ ID No. 1, the nucleotide sequence of the recognition DNA is shown in SEQ ID No. 2, and the nucleotide sequence of the helper DNA is shown in SEQ ID No. 3. During their research, the inventors discovered that using the single-stranded DNA with the nucleotide sequence of SEQ ID No. 1, the recognition DNA with the nucleotide sequence of SEQ ID No. 2, and the helper DNA with the nucleotide sequence of SEQ ID No. 3 to detect ochratoxin A results in higher sensitivity, a wider detection range, and a lower limit of detection.

[0052] SEQ ID No. 1: 5'-CCGATGCTCCCTTTACGC-3'.

[0053] SEQ ID No. 2:

[0054] 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3';

[0055] SEQ ID No. 3:

[0056] 5'-TTTTTTATCGACCGATGCTCCATAGGCGTAAAGGGAGCATCGG-3'.

[0057] Preferably, in step (1), the molar ratio of the recognition DNA to the single-stranded DNA is 1:1.25-1.75, specifically 1:1.25, 1:1.35, 1:1.45, 1:1.55, 1:1.65, 1:1.75, and any value within the range formed by any two of these values. Under this molar ratio, the hybridization effect of the recognition DNA and the single-stranded DNA is better.

[0058] To further improve the hybridization effect of recognizing DNA and single-stranded DNA, preferably, the conditions of reaction I include: a temperature of 90-98℃, specifically 90℃, 92℃, 94℃, 96℃, 98℃, and any value within the range formed by any two of these values; and a time of 3-8 min, specifically 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, and any value within the range formed by any two of these values.

[0059] Preferably, in step (2), the amount of auxiliary DNA added is 1-1.2 mol relative to 1 mol of single-stranded DNA.

[0060] Preferably, in step (2), the conditions for reaction II include a temperature of 30-45°C and a time of 60-90 min; the conditions for reaction III include a temperature of 30-45°C and a time of 20-30 min. Under these conditions, there is a good binding effect between recognition DNA and fungal toxins, as well as between helper DNA and single-stranded DNA.

[0061] Preferably, in step (2), the coenzyme is capable of digesting the single-stranded-helper DNA complex to release the activated DNA and the single-stranded DNA, and is preferably an Exo III enzyme. During their research, the inventors discovered that the single-stranded-helper DNA can release single-stranded DNA under the action of the Exo III enzyme. This single-stranded DNA can continue to react with the helper DNA to form single-stranded-helper DNA, which can then continue to react with the Exo III enzyme, achieving single-stranded DNA recycling and thus improving the utilization rate of single-stranded DNA. Furthermore, under the action of the Exo III enzyme, a large amount of activated DNA that binds to the CRISPR-Cas12a system can be generated, amplifying the trans-cleavage efficiency of CRISPR-Cas12a and increasing the signal intensity generated by the signal probe in the electrochemical biosensor after cleavage. This can effectively increase the detection sensitivity, allowing for quantitative analysis of ochratoxin A in the sample through changes in the electrochemical signal response.

[0062] Specifically, when the reaction mixture II contains the single-stranded DNA, the method further includes: repeating reaction III with the single-stranded DNA that may be present in the reaction mixture II.

[0063] Preferably, in step (2), the reaction IV time is 60-90 min. Under these conditions, the enzymatic digestion reaction is more complete, and the yield of activated DNA is higher.

[0064] The reaction can be terminated using any feasible method disclosed in the prior art. For example, the reaction can be terminated by heating, wherein the heating conditions include a temperature of 80-90°C and a time of 5-15 minutes. This method is simple and convenient, and can reduce damage to other DNA components.

[0065] According to the present invention, in step (3), the CRISPR-Cas12a system solution can be a publicly disclosed system solution. Exemplarily, in step (4), the CRISPR-Cas12a system solution contains crRNA, Cas12a protein, a ribonuclease inhibitor, and a buffer; wherein the nucleotide sequence of the crRNA is shown in SEQ ID No. 5, and the nucleic acid sequence of the activating DNA is shown in SEQ ID No. 4. The Cas12a protein is commercially available or can be synthesized using methods disclosed in the prior art, and the ribonuclease inhibitor is commercially available.

[0066] SEQ ID No.4: 5'-TTTTTTATCGACCGATGCTCCATAG-3';

[0067] SEQ ID No. 5:

[0068] 5'-UAAUUUCUACUAAGUGUAGAUCUAUGGAGCAUCGGUCGAUAAAA-3'.

[0069] According to the present invention, in the CRISPR-Cas12a system solution, the concentration of crRNA is 20-100 nM, the concentration of Cas12a protein is 20-100 nM, and the concentration of ribonuclease inhibitor is 20-60 U / μL. Exemplarily, the total volume after mixing the activated DNA with the CRISPR-Cas12a system solution can be 20 μL. The method for preparing the CRISPR-Cas12a system solution includes: mixing 2 μL of 10*Buffer, 1 μL of ribonuclease inhibitor, 1 μL of crRNA, 1 μL of Cas12a protein, and 5 μL of reaction solution, and finally adding ultrapure water to a final volume of 20 μL.

[0070] Preferably, in step (3), the activation conditions include: a temperature of 30-45°C, specifically 30°C, 35°C, 40°C, 45°C, and any value within the range formed by any two of these values; and a time of 20-30 min, specifically 20 min, 22 min, 24 min, 26 min, 30 min, and any value within the range formed by any two of these values.

[0071] Preferably, in step (4), the conditions for reaction V include: a temperature of 30-45°C, specifically 30°C, 35°C, 40°C, 45°C, and any value within the range formed by any two of these values; and a time of 40-150 min, specifically 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min, 130 min, 140 min, 150 min, and any value within the range formed by any two of these values.

[0072] Preferably, the electrochemical biosensor includes an electrode and a functional layer modified on the electrode, the functional layer containing polyacrylamide hydrochloride, cerium dioxide, and ferrocene-labeled nonspecific single-stranded DNA (Fc-ssDNA).

[0073] According to the present invention, ferrocene in Fc-ssDNA provides an electrical signal during electrochemical detection. When this electrochemical biosensor is applied to the CRISPR-Cas12a system for detecting mycotoxins, the non-specific single-stranded DNA in Fc-ssDNA is used to cleave the activated Cas12a protein to change the electrochemical signal. At the same time, since Fc-ssDNA is coupled with polyacrylamide hydrochloride (PAH) and cerium dioxide (CeO2), the change in electrochemical signal is further amplified. This not only enables quantitative detection of mycotoxins, but also provides high detection sensitivity, low cost, and good specificity.

[0074] The preparation method of the electrochemical biosensor includes the following steps: a modification solution containing polyacrylamide hydrochloride, cerium dioxide, and ferrocene-labeled nonspecific single-stranded DNA is drop-coated onto the surface of an electrode and dried to form a functional layer containing polyacrylamide hydrochloride, cerium dioxide, and ferrocene-labeled nonspecific single-stranded DNA on the surface of the electrode.

[0075] According to the present invention, the modification solution is obtained by mixing a composite solution containing polyacrylamide hydrochloride (PAH) / cerium dioxide (CeO2) with a solution containing Fc-ssDNA in a certain proportion. Exemplarily, the concentration of PAH in the composite solution is 2.5-7.5 mg / mL, the concentration of CeO2 is 1-2 mg / mL, the concentration of Fc-ssDNA in the Fc-ssDNA-containing solution is 2.5-3.5 μM, and the composite solution and the Fc-ssDNA-containing solution are mixed at a volume ratio of 1:1 to obtain the modification solution.

[0076] According to the present invention, the drop-coating of the modifying solution results in the electrode surface being partially or completely coated with the modifying solution. Preferably, the entire contact surface between the electrode and the electrolyte is coated with the modifying solution.

[0077] According to the present invention, the purpose of drying the modified solution after it is drop-coated onto the surface of the electrode is to remove the solvent from the modified solution, so that PAH, CeO2, and Fc-ssDNA in the modified solution adhere and modify the electrode surface to form a functional layer. Preferably, the drying conditions at least satisfy the following: temperature of 20-40°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C, and any value within the range formed by any two of these values; and time of 8-15 hours, for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, and any value within the range formed by any two of these values. Exemplarily, after the modified solution is drop-coated onto the surface of the electrode, it can be dried overnight at room temperature.

[0078] More preferably, the molar ratio of the polyacrylamide hydrochloride, the cerium dioxide, and the ferrocene-labeled nonspecific single-stranded DNA is 1 × 10⁻⁶. 4 -2×10 4 1.5×10 3 -4×10 3 1. The inventors have discovered that, under this preferred embodiment, it is advantageous to better couple Fc-ssDNA with polyacrylamide hydrochloride (PAH) / cerium dioxide (CeO2), thereby enhancing the amplification effect of the electrochemical signal of Fc-ssDNA.

[0079] According to the present invention, the ferrocene-labeled nonspecific single-stranded DNA (Fc-ssDNA) can be modified with ferrocene at its 3' end using any single-stranded DNA sequence that can be cleaved by the activated Cas12a protein, so that the presence of fungal toxins can be characterized by changes in the electrical signal of an electrochemical biosensor. Preferably, the nucleotide sequence of the ferrocene-labeled nonspecific single-stranded DNA is shown in SEQ ID No. 6.

[0080] SEQ ID No. 6: 5'-TTTTTTAAAAAAAAATTTTT-ferrocene-3'.

[0081] The electrode can be any type of electrode capable of measuring electrical signals, such as a glassy carbon electrode, gold electrode, silver electrode, platinum electrode, etc. Preferably, the electrode is a glassy carbon electrode, gold electrode, or platinum electrode. A glassy carbon electrode is preferred. Before use, the glassy carbon electrode needs to be pretreated. The pretreatment method can be a conventional glassy carbon electrode treatment method, specifically: polishing the glassy carbon electrode to a mirror surface with 0.3 mm and 0.5 mm Al2O3 slurry respectively, then rinsing it thoroughly with ultrapure water, and air-drying it for later use. When using a glassy carbon electrode, the amount of modification solution added is 3-8 μL.

[0082] To further improve the accuracy of detection, preferably, the method further includes: before step (4), detecting the initial electrochemical signal of the electrochemical biosensor and calculating the difference between the electrochemical signal and the initial electrochemical signal.

[0083] According to a particularly preferred embodiment of the present invention, a method for detecting ochratoxin A based on the CRISPR-Cas12a system is provided, the detection principle being as follows: Figure 1 As shown, it includes the following steps:

[0084] (1) A single-stranded DNA complex is obtained by reacting a recognition DNA with a molar ratio of 1:1.25-1.75 as shown in SEQ ID No.2 and a single-stranded DNA with a molar ratio of 1:1.25-1.75 with a single-stranded DNA with a molar ratio of SEQ ID No.1 at a temperature of 90-98℃ for 3-8 min.

[0085] (2) After reacting the single-stranded recognition DNA complex and the test sample that may contain fungal toxins at a temperature of 30-45℃ for 60-90 min, reacting it with the helper DNA with the nucleotide sequence shown in SEQ ID No.3 at a temperature of 30-45℃ for 30-60 min to obtain reaction mixture I, mixing reaction mixture I with Exo III enzyme and reacting for 60-90 min, then heating the temperature to 80-90℃ and holding for 5-15 min to obtain reaction mixture II containing the activated DNA;

[0086] (3) The reaction mixture II is mixed with the CRISPR-Cas12a system solution at a temperature of 30-45℃ and activated for 20-30 min to obtain the reaction solution;

[0087] The CRISPR-Cas12a system solution contains crRNA, Cas12a protein, ribonuclease inhibitor, and buffer; wherein, in the CRISPR-Cas12a system solution, the concentration of crRNA is 20-100 nM, the concentration of Cas12a protein is 20-100 nM, the concentration of ribonuclease inhibitor is 20-60 U / μL, the nucleotide sequence of the crRNA is shown in SEQ ID No. 5, and the nucleic acid sequence of the activating DNA is shown in SEQ ID No. 4;

[0088] (4) The electrochemical biosensor was subjected to differential pulse voltammetry detection in a detection solution with a concentration of 0.05-0.15 mol / L Tris-HCl buffer (containing 0.1-0.3 mol / L NaCl) and a pH of approximately 7.4, and its peak current I0 was detected.

[0089] The reaction solution obtained in step (3) is added to the surface of the electrochemical biosensor and reacted at a temperature of 30-45℃ for 40-150 min. After the reaction is completed, the electrochemical biosensor is subjected to differential pulse voltammetry detection to detect its peak current I.

[0090] (5) Calculate the peak change rate according to ΔI(%)=[(I0-I) / I0]×100%, and calculate the amount of OTA in the sample to be tested according to the standard curve.

[0091] Depend on Figure 1 As can be seen, the method provided by this invention, in the presence of OTA, OTA is recognized by the recognition DNA (aDNA) in the single-stranded recognition DNA complex (aDNA-cDNA), thereby releasing single-stranded DNA (cDNA). The released cDNA can hybridize with the designed helper DNA (HP) to form a single-stranded helper DNA complex (cDNA-HP). The introduced Exo III enzyme then digests the formed cDNA-HP along the 3' end of HP, leaving only a portion of the HP sequence (tDNA portion), and releasing the complete cDNA. The cDNA can continue to react with HP in the system to form a single-stranded helper DNA complex, thus achieving cDNA cycling, and can also generate a large amount of activating DNA (tDNA). The tDNA can further bind to crRNA to activate the trans-cleavage activity of Cas12a. When the activated CRISPR-Cas12a system is introduced into the modified electrode surface, a large amount of Fc-ssDNA will be cleaved, causing a sharp decrease in the ferrocene electrochemical signal, resulting in a signal value lower than the initial electrochemical signal of the electrochemical biosensor during detection. Figure 1 B). Therefore, quantitative detection of OTA can be achieved by recording the changes in electrochemical signals at different concentrations of OTA using an electrochemical workstation.

[0092] The method described above, which utilizes the preferred embodiment, amplifies changes in the electrochemical signal, significantly improving measurement accuracy, detection range, and sensitivity, while lowering the detection limit. Simultaneously, it reduces the cost of ochratoxin A detection methods and improves detection specificity.

[0093] The present invention will be described in detail below through examples. In the following examples, polyacrylamide hydrochloride was purchased from Aladdin, product number 71550-12-4, with a relative molecular mass of 93.56; tris(hydroxymethyl)aminomethane (Tris), ochratoxin A (OTA), ochratoxin B (OTB), vomitoxin (DON), zearalenone (ZEN), fumonisin (FB1), T-2 toxin, and aflatoxin B1 (AFB1) were all purchased from Aladdin Industrial Corporation (Shanghai, China); Cas12a protein was purchased from Genscript Biotech Inc.; Exo III and all DNA sequences in this invention were ordered from Shanghai Sangon Biotech Co., Ltd.; glassy carbon electrodes and electrochemical workstations were from Shanghai Chenhua Instrument Co., Ltd.; other raw materials and reagents were conventional commercially available products. Polyacrylamide gel electrophoresis measurements were performed according to the method described in the document (Zhang DC, Yan YR, Que HY, Yang, TT, Cheng, XX, Ding SJ, Zhang XM, Cheng W. CRISPR / Cas12a-Mediated Interfacial Cleaning of Hairpin DNA Reporter for Electrochemical Nucleic Acid Sensing, ACSSens. 2020, 5(2), 557-562).

[0094] In the following examples, unless otherwise specified, room temperature refers to 25±5℃, and one night refers to 8-12 hours;

[0095] The detection solution of the electrochemical biosensor is a 0.1 mol / L Tris-HCl buffer solution (containing 0.2 mol / L NaCl) with a pH of 7.4; the differential pulse detection range is -0.2V to 0.7V; ΔI% is calculated using the following formula: ΔI% = [(I0-I) / I0] × 100%, where I0 is the initial peak current of the electrochemical biosensor; and I is the peak current of the electrochemical biosensor after treatment with the reaction solution.

[0096] Example 1

[0097] Fabrication of electrochemical biosensors:

[0098] (1) A modified solution was prepared by mixing a composite solution containing polyacrylamide hydrochloride (PAH) and cerium dioxide (CeO2) with a solution containing 3 μM Fc-ssDNA at a volume ratio of 1:1. The concentration of CeO2 in the composite solution was 1.5 mg / mL, the concentration of PAH was 5 mg / mL, and the nucleotide sequence of Fc-ssDNA was shown in SEQ ID No. 6.

[0099] (2) 5 μL of the modification solution was dropped onto the surface of the treated glassy carbon electrode and left to dry overnight at room temperature to obtain an electrochemical biosensor (PAH-CeO2&Fc-ssDNA / GCE).

[0100] Differential pulse voltammetry was performed on the electrochemical biosensor in a detection solution containing 0.1 mol / L Tris-HCl buffer (containing 0.2 mol / L NaCl) and a pH of approximately 7.4. The resulting CV curve is shown below. Figure 2 As shown by curve b in the middle.

[0101] Example 2

[0102] Fabrication of electrochemical biosensors:

[0103] (1) A modified solution was prepared by mixing a composite solution containing polyacrylamide hydrochloride (PAH) and cerium dioxide (CeO2) with a 3 μM Fc-ssDNA solution at a volume ratio of 1:1. The concentrations of CeO2 in the composite solution were 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL and 3 mg / mL, and the concentrations of PAH were 5 mg / mL. The nucleotide sequence of Fc-ssDNA was shown in SEQ ID No. 6.

[0104] (2) 5 μL of the modification solution was drop-coated onto the surface of the treated glassy carbon electrode and left to dry overnight at room temperature to obtain an electrochemical biosensor.

[0105] Methods for testing OTA:

[0106] (1) 5 μL of 2 mol / L recognition DNA (aDNA) with nucleotide sequence as shown in SEQ ID No. 2 and 5 μL of 3 mol / L single-stranded DNA (cDNA) with nucleotide sequence as shown in SEQ ID No. 1 were reacted at 95 °C for 5 min and cooled to room temperature to obtain single-stranded recognition DNA complex.

[0107] (2) The single-stranded recognition DNA complex and 5 μL of 1 ng / mL OTA solution were reacted at 37°C for 75 min to release the single-stranded DNA. Then, 5 μL of 6 mol / L auxiliary DNA (HP) with the nucleotide sequence shown in SEQ ID No.3 was added and reacted at 37°C for 45 min to obtain reaction mixture I containing the single-stranded auxiliary DNA complex (cDNA-HP).

[0108] (3) Mix the reaction mixture I with 5 μL of 8 U / μL Exo III enzyme and react for 75 min. Then heat the temperature to 85 °C and keep it for 10 min to obtain the reaction mixture II containing the activated DNA (tDNA).

[0109] (4) The reaction mixture II was mixed with 15 μL of CRISPR-Cas12a system solution and activated for 30 min at a temperature of 30-45℃ to obtain the reaction solution;

[0110] The CRISPR-Cas12a system solution contains crRNA, Cas12a protein, ribonuclease inhibitor, and buffer; wherein the concentration of crRNA is 60 nM, the concentration of Cas12a protein is 60 nM, the concentration of ribonuclease inhibitor is 40 U / μL, the buffer solution has a pH of 7.4 and contains 1.0 mol / L NaCl, 0.5 mol / L Tris, 0.1 mol / L MgCl2, and 1 mg / mL bovine serum albumin; the nucleotide sequence of the crRNA is shown in SEQ ID No. 5, and the nucleic acid sequence of the activated DNA is shown in SEQ ID No. 4.

[0111] (5) The electrochemical biosensor was subjected to differential pulse voltammetry detection in a detection solution with a concentration of 0.1 mol / L Tris-HCl buffer (containing 0.2 mol / L NaCl) and a pH of approximately 7.4, and its peak current I0 was detected.

[0112] Take 5 μL of the reaction solution obtained in step (4) and drop it onto the surface of the electrochemical biosensor. React at 37 °C for 90 min. After the reaction is completed, perform differential pulse voltammetry detection on the electrochemical biosensor in a detection solution with a concentration of 0.1 mol / L Tris-HCl buffer (containing 0.2 mol / L NaCl) and a pH of about 7.4 to detect its peak current I.

[0113] (6) Calculate the peak change rate according to ΔI(%) = [(I0-I) / I0] × 100%, and the resulting ΔI(%) change graph is shown below. Figure 4 As shown in (A).

[0114] The results showed that as the CeO2 concentration increased from 0.5 to 3.0 mg / mL, the percentage change in current (ΔI) reached its maximum value at a CeO2 concentration of 1.5 mg / mL, and the preferred CeO2 concentration was 1-2 mg / mL.

[0115] Example 3

[0116] The electrochemical biosensor was prepared according to the method described in Example 2, except that in step (1), the concentration of CeO2 in the composite solution was 1.5 mg / mL, and the concentrations of PAH were 1 mg / mL, 3 mg / mL, 5 mg / mL, 7 mg / mL, and 8 mg / mL, respectively.

[0117] The method described in Example 2 is used to detect OTA, except that in step (6), the peak change rate is calculated according to ΔI(%) = [(I0-I) / I0] × 100%, and the resulting ΔI(%) change graph is shown below. Figure 4 As shown in (B).

[0118] The results showed that ΔI(%) increased with increasing PAH concentration, reaching a maximum at a PAH concentration of 5.0 mg / mL, and the preferred PAH concentration was 3-5 mg / mL.

[0119] Example 4

[0120] The electrochemical biosensor was prepared according to the method described in Example 2, except that in step (1), the concentration of CeO2 in the composite solution was 1.5 mg / mL, and the concentrations of Fc-ssDNA were 1 μM, 2 μM, 3 μM, 4 μM, and 5 μM, respectively.

[0121] The method described in Example 2 is used to detect OTA, except that in step (6), the peak change rate is calculated according to ΔI(%) = [(I0-I) / I0] × 100%, and the resulting ΔI(%) change graph is shown below. Figure 4 As shown in (C).

[0122] The results showed that the percentage change in current ΔI (%) gradually increased with the concentration of Fc-ssDNA in the range of 1-3 μM, and reached the maximum at 3 μM, indicating that the Fc-ssDNA probe had reached saturation on the electrode surface. The preferred concentration of Fc-ssDNA was 3-5 μM.

[0123] Example 5

[0124] Electrochemical biosensors were prepared according to the method described in Example 2.

[0125] OTA was detected according to the method described in Example 2, except that in step (4), the Cas12a concentration was 20, 40, 60, 80, and 100 nM, and the resulting ΔI (%) change graph is shown below. Figure 4 As shown in (D).

[0126] As the concentration of Cas12a increases, ΔI (%) also increases, reaching its maximum value when the concentration of Cas12a is 60 nM. The preferred concentration of Cas12a is 40-80 nM, indicating that the concentration of Cas12a has a significant impact on the response of Fc-ssDNA probe signal value.

[0127] Example 6

[0128] Electrochemical biosensors were prepared according to the method described in Example 2.

[0129] OTA was detected according to the method described in Example 2, except that in step (4), the crRNA concentration was 20, 40, 60, 80, and 100 nM, and the resulting ΔI (%) change graph is shown below. Figure 4 As shown in (E).

[0130] As the concentration of crRNA increases, ΔI (%) also increases, reaching its maximum value when the crRNA concentration is 60 nM. The preferred concentration of crRNA is 40-80 nM, indicating that the concentration of crRNA has a significant impact on the response of Fc-ssDNA probe signal value.

[0131] Example 7

[0132] Electrochemical biosensors were prepared according to the method described in Example 2.

[0133] OTA was detected according to the method described in Example 2, except that in step (5), the reaction time was 10, 30, 60, 90, 120, and 150 min, and the resulting ΔI (%) change graph is shown below. Figure 4 As shown in (F).

[0134] ΔI (%) increases rapidly before 90 min, and then increases slightly with time. The preferred activated CRISPR-Cas12 system has a cutting time of 60-90 min on the electrode surface.

[0135] Example 8

[0136] An electrochemical biosensor was prepared according to the method described in Example 2, except that the CeO2 concentration was 1.5 mg / mL.

[0137] OTA was detected according to the method described in Example 2, except that in step (2), the concentration of OTA was 0 ng / mL and 1×10⁻⁶.-6 ng / mL, 1×10 -5 ng / mL, 1×10 -4 ng / mL, 1×10 -3 ng / mL, 1×10 -2 ng / mL, 0.1 ng / mL, 1 ng / mL, 5 ng / mL; the resulting DPV graph is shown below. Figure 5 As shown in (A), the resulting graph of ΔI (%) change is as follows: Figure 5 As shown in (B).

[0138] In 1×10 -6 Within the range of 5.0 ng / mL, the peak current signal of ferrocene decreases with increasing OTA. Figure 5 The results in (B) indicate that the percentage change in ferrocene current ΔI(%) is linearly correlated with the logarithm of OTA concentration within this range, and the regression equation is ΔI(%) = 3.95logOTA + 86.99(R). 2 =0.982), the lowest detection limit of the electrochemical sensing detection method for ochratoxin A constructed in this invention is 0.74 fg / mL.

[0139] Example 9

[0140] Electrochemical biosensors were prepared according to the method described in Example 2.

[0141] OTA was detected according to the method described in Example 2, except that OTA was replaced with substances such as ochratoxin B (OTB), vomitoxin (DON), zearalenone (ZEN), fumonisin (FB), T-2 toxin (T-2), and aflatoxin B1 (AFB1), or no OTA was added. The resulting ΔI (%) change graph is shown in the figure. Figure 6 As shown;

[0142] The results showed that only very low percentage changes in current were observed in the presence of ochratoxin B (OTB), vomitoxin (DON), zearalenone (ZEN), fumonisin (FB1), T-2 toxin, and aflatoxin B1 (AFB1), but significant percentage changes in current were observed in the presence of a mixed solution containing multiple mycotoxins including OTA. This demonstrates that the recognition DNA with the nucleotide sequence shown in SEQ ID No. 2 has high specificity in detecting OTA.

[0143] Comparative Example 1

[0144] An electrochemical biosensor was prepared according to the method described in Example 1, except that in step (1), the composite solution did not contain cerium dioxide, and the electrochemical biosensor (PAH&Fc-ssDNA / GCE) was obtained.

[0145] Differential pulse voltammetry was performed on the electrochemical biosensor (PAH&Fc-ssDNA / GCE), and the resulting CV curve is shown below. Figure 2 As shown by curve a in the middle.

[0146] Test Example 1

[0147] pass Figure 2 The data show that the modified electrodes provided in Example 1 and Comparative Example 1 both exhibit a set of redox peaks, but the redox peak of curve b is significantly better than that of curve a. This indicates that PAH-CeO2 & Fc-ssDNA / GCE has higher electron transfer efficiency. Therefore, the combination of CeO2 nanorods and PAH can not only enhance the immobilization of Fc-ssDNA, but also improve the electron transfer efficiency.

[0148] Test Example 2

[0149] The reaction solution in Example 2 and the CRISPR-Cas12a system were measured by polyacrylamide gel electrophoresis, and the results are shown in the figure below. Figure 3 As shown. In Figure 3 In lane A, M is a 25-500 bp DNA marker. The band in lane 4 (cDNA-aDNA) is significantly higher than the cDNA band (lane 1) and the aDNA band (lane 2), indicating that cDNA and aDNA hybridize to form a new dsDNA band. In lane 5, when dsDNA (cDNA-aDNA) is incubated with OTA, a band clearly belonging to cDNA is observed compared to lane 1, indicating that OTA successfully binds to the aDNA in the dsDNA and releases the cDNA. After the addition of HP and Exo III, the released cDNA hybridizes with HP to form another new dsDNA band (lane 6). This dsDNA is then gradually digested by Exo III from the 3' blunt end of HP, releasing the complete cDNA and leaving a short ssDNA fragment (tDNA). The released cDNA can reopen the hairpin structure of additional HP and hybridize with it, triggering the Exo III helper cycle reaction and producing a large tDNA. Therefore, a bright, short ssDNA (tDNA) band was observed above the cDNA band in lane 7. This proves that the Exo III enzyme digestion reaction did indeed produce a large amount of tDNA.

[0150] The cleavage activity verification results of the CRISPR-Cas12a system are as follows: Figure 3As shown in Figure B, Fc-ssDNA was added to each lane. Bands for tDNA and Fc-ssDNA were clearly observed in lane 1. When only Cas12a (lane 2) or crRNA was present (lane 3), the bands for tDNA and Fc-ssDNA remained unchanged. This is because the Cas12a-crRNA double-stranded complex did not form in the absence of Cas12a or crRNA, and therefore the recognition and cleavage reaction did not occur. Similarly, in the absence of tDNA (lane 4), the Fc-ssDNA band remained unchanged because the Cas12a-crRNA complex is inactive in the absence of tDNA. Only in lane 5, when Cas12a, crRNA, and tDNA were all present, was arbitrary cleavage of Fc-ssDNA by Cas12a triggered, leading to the disappearance of the crRNA, tDNA, and Fc-ssDNA bands. These results demonstrate the feasibility of the designed electrochemical biosensor for OTA detection.

[0151] 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 inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for detecting mycotoxins, characterized in that, Includes the following steps: (1) Reaction I is performed between single-stranded DNA and the recognition DNA of fungal toxin to obtain a single-stranded-recognition DNA complex, wherein the single-stranded DNA is DNA complementary to the recognition DNA; (2) After mixing the single-stranded recognition DNA complex with the test sample that may contain fungal toxins for reaction II, it is mixed with helper DNA containing the activating DNA fragment for reaction III to obtain reaction mixture I. The reaction mixture I is mixed with a helper enzyme for reaction IV to obtain reaction mixture II that may contain the activating DNA. The helper DNA is DNA complementary to the single-stranded DNA. (3) The reaction mixture II is mixed with the CRISPR-Cas12a system solution for activation to obtain the reaction solution; (4) The reaction solution obtained in step (3) is added to the electrochemical biosensor for reaction V. After the reaction V is completed, the electrochemical signal of the electrochemical biosensor is detected. The helper DNA can bind to the single-stranded DNA to form a single-stranded helper DNA complex, and the helper enzyme can digest the single-stranded helper DNA complex to release the activated DNA.

2. The method according to claim 1, characterized in that, The fungal toxin is a mycotoxin; The mycotoxin is ochratoxin A, the nucleotide sequence of the single-stranded DNA is shown in SEQ ID No. 1, the nucleotide sequence of the recognition DNA is shown in SEQ ID No. 2, and the nucleotide sequence of the helper DNA is shown in SEQ ID No.

3.

3. The method according to claim 1 or 2, characterized in that, In step (1), the molar ratio of the recognition DNA to the single-stranded DNA is 1:1.25-1.75; The conditions for reaction I include: a temperature of 90-98℃ and a time of 3-8 min.

4. The method according to claim 1 or 2, characterized in that, In step (2), the amount of auxiliary DNA added is 1-1.2 mol relative to 1 mol of single-stranded DNA; The conditions for reaction II include: a temperature of 30-45°C and a time of 60-90 min; The conditions for reaction III include: a temperature of 30-45°C and a time of 30-60 min.

5. The method according to claim 1 or 2, characterized in that, In step (2), the coenzyme digests the single-stranded helper DNA complex to release the activated DNA and the single-stranded DNA, and the coenzyme is an Exo III enzyme.

6. The method according to claim 5, characterized in that, When the reaction mixture II contains the single-stranded DNA, the method further includes: repeating reaction III with the single-stranded DNA that may be present in the reaction mixture II.

7. The method according to claim 1 or 2, characterized in that, In step (3), the CRISPR-Cas12a system solution contains crRNA, Cas12a protein, ribonuclease inhibitor and buffer; wherein the nucleotide sequence of the crRNA is shown in SEQ ID No. 5, and the nucleic acid sequence of the activated DNA is shown in SEQ ID No.

4.

8. The method according to claim 1 or 2, characterized in that, In step (3), the activation conditions include a temperature of 30-45°C and a time of 20-30 min.

9. The method according to claim 1 or 2, characterized in that, The electrochemical biosensor includes an electrode and a functional layer modified on the electrode, the functional layer containing polyacrylamide hydrochloride, cerium dioxide, and ferrocene-labeled nonspecific single-stranded DNA. The molar ratio of the polyacrylamide hydrochloride, the cerium dioxide, and the ferrocene-labeled nonspecific single-stranded DNA is 1 × 10⁻⁶. 4 -2×10 4 1.5×10 3 -4×10 3 :1; The nucleotide sequence of the nonspecific single-stranded DNA is shown in SEQ ID No. 6; The electrode is a glassy carbon electrode, a gold electrode, or a platinum electrode.

10. The method according to claim 1 or 2, characterized in that, The method further includes: before step (4), detecting the initial electrochemical signal of the electrochemical biosensor and calculating the percentage difference between the electrochemical signal and the initial electrochemical signal.

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