Electrochemiluminescence ochratoxin A sensing platform based on enzyme-free catalysis DNA nano walker, preparation method and application

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to an electrochemical luminescence ochratoxin A sensing platform based on an enzyme-free catalysis DNA nano walker, a preparation method and application. According to the invention, the gold nanocluster is loaded on the silver nanomaterial for the first time to realize high electrochemical luminescence performance, the stability and sensitivity of an ochratoxin A sensing platform are improved, and meanwhile, an enzyme-catalysis-free nano walker is designed for amplifying DNA to realize specific analysis of low-concentration ochratoxin A.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry, specifically relating to an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker, its preparation method, and its application. Background Technology

[0002] Ochratoxin A is a highly toxic fungal toxin that primarily damages the kidneys and liver, and may also cause nervous system damage, immune system abnormalities, and even has potential carcinogenic, teratogenic, and mutagenic effects. Therefore, accurate and rapid detection can promptly identify the presence of this toxin in food, thereby preventing the consumption of contaminated food and reducing the risk of poisoning. In recent years, electrochemiluminescence sensing platforms have been widely used in the field of food safety testing due to their advantages such as rapid analysis speed, high sensitivity, and wide linear range. Summary of the Invention

[0003] This invention provides an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker, its preparation method, and its applications. The invention also provides a method for preparing this sensing platform and a method for detecting ochratoxin A based on this platform.

[0004] Technical solution: A method for preparing an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker, comprising the following steps:

[0005] Step 1: Preparation of DNA nanowalkers: DNA1 and DNA2 are incubated to form double strands. Carboxyl-modified magnetic beads are activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and then incubated with the double strands and N-hydroxysuccinimide to form windmill-structured nanowalkers.

[0006] Step 2: Polish the glassy carbon electrode with alumina polishing powder;

[0007] Step 3: Prepare glassy carbon electrodes modified with gold and silver nanomaterials;

[0008] Step 4: Preparation of an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker: First, mix ochratoxin A aptamer DNA with DNA3 and incubate for a period of time. Then, add ochratoxin A solution and incubate for a period of time to obtain a mixed solution. Drop the mixed solution onto the DNA nanowalker prepared in Step 1 and incubate for a period of time. Then, add DNA4 and continue incubating for a period of time. Remove the magnetic beads, drop the supernatant onto the glassy carbon electrode obtained in Step 3, dry it, and wash it with PBS buffer solution to obtain the electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker.

[0009] The sequence of DNA1 is shown in SEQ ID No. 1, the sequence of DNA2 is shown in SEQ ID No. 2, the sequence of DNA3 is shown in SEQ ID No. 3, the sequence of DNA4 is shown in SEQ ID No. 4, and the sequence of ochratoxin A aptamer DNA is shown in SEQ ID No. 5.

[0010] The 5' end of DNA1 is modified with an amino group; the 5' end of DNA2 is covalently linked to a thiol group via a hexamethylene chain, and the 3' end is modified with the electrochemiluminescent quencher MB.

[0011] Furthermore, step one specifically involves the following steps:

[0012] (1) Incubate 19 µL~22 µL of DNA1 (5-15 μM) and 19 µL~22 µL of DNA2 (5-15 μM) at 37 °C for 20 minutes to form double strands;

[0013] (2) Activate the aqueous solution of 5 mg / mL, 19 ~ 22 µL of carboxyl-modified magnetic beads with 0.1 ~ 0.3 mol / L, 40 µL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for 30 minutes;

[0014] (3) The activated magnetic beads, 20 µL of double strands and 0.1 ~ 0.3 mol / L, 40 µL of N-hydroxysuccinimide were incubated together for 12 hours to form a windmill-structured nanowalker.

[0015] Furthermore, the specific operation of step two is as follows:

[0016] A glassy carbon electrode with a diameter of 4 mm was polished sequentially with alumina polishing powders of 1.0 μm, 0.3 μm, and 0.05 μm, respectively, and then rinsed with ultrapure water to obtain a smooth glassy carbon electrode.

[0017] Furthermore, in step three, the preparation method of the glassy carbon electrode modified with gold and silver nanomaterials is as follows:

[0018] 8-11 µL of an aqueous solution of gold and silver nanomaterials at a concentration of 1-3 mg / mL was drop-coated onto the surface of the glassy carbon electrode obtained in step two and then dried at room temperature to obtain a glassy carbon electrode modified with gold and silver nanomaterials.

[0019] Furthermore, in step three, the preparation method of the gold and silver nanomaterials is as follows:

[0020] S1. Add sodium borohydride to silver nitrate solution, place in a water bath at 59-62°C for 5-6 hours, centrifuge and wash with deionized water to obtain silver nanomaterials, disperse the silver nanomaterials in water for later use.

[0021] S2. Chloroauric acid solution was stirred thoroughly with 4,6-diamino-2-thiopyrimidine for 1 h, left to stand in the dark for 1 day, mixed with silver nanomaterials and citric acid, reacted for 3 days, centrifuged and washed, and collected to obtain gold and silver nanomaterials.

[0022] Furthermore, in step three, the preparation method of the gold and silver nanomaterials is as follows:

[0023] (1) Add 0.34 g of sodium borohydride to 420 mL of 0.1 mol / L silver nitrate solution, stir thoroughly, and then place in a water bath at 59 ~ 62 ℃ for 5 ~ 6 hours. The prepared sample is washed 12 times with deionized water by centrifugation to obtain silver nanomaterials. Disperse the silver nanomaterials in 20 mL of water for subsequent use.

[0024] (2) Stir 0.5-1 mL of 1% chloroauric acid solution (water as solvent) and 0.5-1 mL of 4,6-diamino-2-thiopyrimidine solution (70 mM, water as solvent) thoroughly for 1 h, let stand in the dark for 1 day, mix the above solution with 0.5-1 mL of silver nanomaterial aqueous solution and 110-120 µL of citric acid solution (12 mM, water as solvent), react for 3 days, centrifuge and wash, collect, and obtain gold and silver nanomaterials.

[0025] Furthermore, the specific operation of step four is as follows:

[0026] (1) First, mix 10 μM, 19 ~ 22 μL of ochratoxin A aptamer DNA with 10 μM, 19 ~ 22 μL of DNA3 and incubate at 37 °C for 30 minutes; then, add 19 ~ 22 μL of ochratoxin A solution with a concentration of 10 pg / mL to 200 ng / mL and incubate at 37 °C for 30 minutes to obtain a mixed solution;

[0027] (2) Add the mixed solution to the nanowalker prepared in step one and incubate for 10 minutes, then add 10 μM and 19 ~ 22 μL DNA4 and continue incubating for 60-80 minutes;

[0028] (3) Remove the magnetic beads, add the supernatant to the glassy carbon electrode obtained in step 3, dry it, and then rinse it with PBS buffer solution with pH 7.0 ~ 8.2 to obtain an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker.

[0029] The present invention also provides a method for detecting ochratoxin A based on the aforementioned sensing platform, the method being as follows:

[0030] (1) Electrochemiluminescence system setup;

[0031] The test was conducted using a three-electrode system, specifically configured as follows: an Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker as the working electrode.

[0032] During the experiment, the electrochemical workstation was used in conjunction with the chemiluminescence detector;

[0033] (2) The intensity of the electrochemiluminescence signal generated by ochratoxin A standard solution of different concentrations was detected by an electrochemiluminescence system, and the working curve was plotted.

[0034] (3) The test sample solution is used instead of the ochratoxin A standard solution for determination, and the concentration of ochratoxin A in the test sample solution is obtained according to the working curve.

[0035] Furthermore, in the method for detecting ochratoxin A based on the aforementioned sensing platform:

[0036] The electrochemical workstation was used in conjunction with a chemiluminescence detector, with the following parameters set: the high voltage of the photomultiplier tube was adjusted to 500~800 V, the scanning potential range of the cyclic voltammetry was set to 0 ~ -1.4 V, and the scanning rate was controlled at 0.2 ~ 0.7 V / s;

[0037] To plot the working curve, the electrochemiluminescence signal intensity generated by different concentrations of ochratoxin A standard solution was detected in 10 mL of PBS buffer solution containing 78 mmol / L to 80 mmol / L potassium persulfate at pH 7.0 to 8.2 using an electrochemiluminescence system, and the working curve was plotted.

[0038] Beneficial effects:

[0039] (1) This invention is the first to combine enzyme-free catalysis and DNA nanowalkers for the detection of ochratoxin A. The enzyme-free sensor has higher stability and is not limited by the problem of easy enzyme inactivation, and can work under a wider range of environmental conditions; it is less expensive, avoiding complex and costly steps such as enzyme extraction, purification and immobilization; it has a faster response speed and shorter detection time. The cyclic amplification strategy combined with DNA nanowalkers enables the detection of lower concentrations of ochratoxin A.

[0040] (2) In this invention, gold nanoclusters are loaded onto silver nanomaterials to suppress non-radiative diffraction by utilizing the framework properties of silver. At the same time, the material has the ability to catalyze electrochemical reactions, thereby enhancing the stability and luminescence intensity of the material. Using gold nanoclusters loaded onto silver nanomaterials as a luminescent agent in an electrochemiluminescent ochratoxin A sensing platform can greatly improve the sensitivity and stability of the sensing platform.

[0041] (3) This invention applies an enzyme-free catalytic DNA nanowalker to the electrochemiluminescence detection of ochratoxin A specific marker recognition and signal amplification. The constructed enzyme-free catalytic DNA nanowalker-based electrochemiluminescence ochratoxin A sensing platform can be applied to the sensitive detection of ochratoxin A. This detection method has the following advantages: simple operation, fast detection speed, wide signal linear range (from 10 pg / mL to 200 ng / mL), and extremely low detection limit (as low as 3.23 pg / mL). Attached Figure Description

[0042] Figure 1 Scanning electron microscope images of silver nanomaterials obtained based on Example 1 and silver nanomaterials loaded with gold nanoclusters.

[0043] Figure 2 The operating curve of the electrochemiluminescent ochratoxin A sensing platform obtained in Example 3 is: I = 13289 - 1954 lg c, R 2 =99.8%; linear range from 10 pg / mL to 200 ng / mL; LOD = 3.23 pg / mL (S / N = 3). Detailed Implementation

[0044] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0045] Example 1

[0046] A method for preparing gold and silver nanomaterials:

[0047] (1) Add 0.34 g of sodium borohydride to 420 mL of 0.1 mol / L silver nitrate solution, stir thoroughly, and then place in a water bath at 60 °C for 6 hours. The prepared sample is washed 12 times with deionized water by centrifugation. Finally, disperse the silver nanomaterials in 20 mL of water for subsequent use.

[0048] (2) 1 mL of 1% chloroauric acid solution (solvent is water) and 1 mL of 4,6-diamino-2-thiopyrimidine (70 mM, solvent is water) were stirred thoroughly for 1 h and allowed to stand in the dark for 1 day. The above solution was then mixed with 1 mL of silver nanomaterial and 120 µL of citric acid (12 mM, solvent is water) and reacted for 3 days. After centrifugation and washing, the gold and silver nanomaterials were collected.

[0049] Figure 1 The images show scanning electron microscope (SEM) images of silver and gold-silver nanomaterials obtained in Example 1. The left image shows the SEM image of the silver nanomaterial, which can be seen to have an overall porous aerogel structure. When gold nanoclusters are loaded onto the silver nanomaterial, as shown... Figure 1 As shown in the right figure, it can be clearly seen that a layer of material is loaded on the silver nanomaterial and the pores are also filled, indicating that the gold nanoclusters have been successfully loaded onto the silver nanomaterial to form gold-silver nanomaterials.

[0050] Example 2

[0051] An electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker is characterized by the following preparation steps:

[0052] Step (1): Fabrication of DNA nanowalkers

[0053] Double-stranded DNA1 / DNA2 was formed by incubating 20 µL of 10 µM DNA2 and 20 µL of 10 µM DNA2 at 37 °C for 20 min. 20 µL of carboxyl-modified magnetic bead solution (5 mg / mL, aqueous solution) was activated with 0.1 mol / L, 40 µL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for 30 min, and then incubated with 20 µL of double-stranded DNA1 / DNA2 and 0.1 mol / L, 40 µL of N-hydroxysuccinimide (NHS) for 12 h to form a windmill-structured nanowalker.

[0054] The sequence of DNA1 (5' to 3', SEQ ID No. 1): NH2-TTT TTT TAT GGC ACA CAC ACA TTTCCG TTG GAT CGG GTG TGG GTG GCG TAA AGG GAG CGC CAC CCA CAC. The 5' end of DNA1 is modified with an amino group (-NH2).

[0055] The sequence of DNA2 (5' to 3', SEQ ID No. 2): SH-(CH2)6-CAA CGG AAA TGT GTG TGT GCCATA-MB. The 5' end of DNA2 is covalently linked to a thiol group (-SH) via a hexamethylene chain, and the 3' end is modified with the electrochemiluminescent quencher MB (methylene blue).

[0056] DNA1 and DNA2 were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0057] Step (2): Polish the glassy carbon electrode with a diameter of 4 mm in sequence with alumina polishing powder of 1.0 µm, 0.3 µm and 0.05 µm until the interface is as smooth as a mirror. Rinse it with ultrapure water to obtain a smooth glassy carbon electrode.

[0058] Step (3): Drop 8 µL of 2 mg / mL gold and silver nanomaterial aqueous solution onto the surface of the glassy carbon electrode polished in step (2), and air dry at room temperature to obtain a glassy carbon electrode modified with gold and silver nanomaterials.

[0059] Step (4): Mix 20 µL of 10 μM ochratoxin A aptamer and 20 µL of 10 μM DNA3 and incubate at 37 °C for 30 minutes; then add 20 µL of 1 ng / mL ochratoxin A solution, mix and incubate at 37 °C for 30 minutes to obtain a mixed solution;

[0060] The above mixed solution was added dropwise to the nanowalker prepared in step (1) and incubated for 10 minutes. 20 µL of 10 μM DNA4 was added and incubated for another 70 minutes. After removing the magnetic beads, the supernatant was added dropwise to the electrode in step (3). After drying, it was rinsed with PBS buffer solution at pH 7.4 to obtain an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker.

[0061] The carboxyl-modified magnetic beads were purchased from Xianfeng Nanomaterials Technology Co., Ltd., and their particle size is 100-200nm. The concentration of the carboxyl-modified magnetic bead solution is 5mg / mL, and the solvent is water. The ochratoxin A solution is a standard solution or a sample solution.

[0062] The sequence of DNA 3 ((5' to 3', SEQ ID No. 3) is: CCTTTACGCCACCCACACCCGATC.

[0063] The sequence of DNA 4 ((5' to 3', SEQ ID No. 4) is: TTACGCCACCCACACCCGATCCAACGGAAATGTGTGTGTGCCATA.

[0064] Ochratoxin A aptamer refers to a DNA sequence that specifically binds to ochratoxin A, and its sequence (5' to 3', SEQ ID No. 5) is: GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA.

[0065] DNA 3, DNA 4, and ochratoxin A aptamer were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0066] Example 3

[0067] An application of an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker is characterized by the following steps:

[0068] (1) Electrochemiluminescence detection of ochratoxin A was performed using a three-electrode system. The specific configuration was as follows: an Ag / AgCl electrode was used as the reference electrode, a platinum wire electrode was used as the counter electrode, and an electrochemiluminescence sensing platform based on an enzyme-free catalytic DNA nanowalker was used as the working electrode. During the experiment, the electrochemical workstation was connected to the chemiluminescence detector, the high voltage of the photomultiplier tube was adjusted to 600 V, the scanning potential range of the cyclic voltammetry was set to 0 ~ -1.4 V, and the scanning rate was controlled at 0.5 V / s.

[0069] (2) In 10 mL of PBS buffer solution containing 80 mmol / L potassium persulfate at pH 7.4, the electrochemiluminescence signal intensity generated by different concentrations of ochratoxin A standard solution was detected using an electrochemiluminescence system, and a working curve was plotted. The electrochemiluminescence signal of the test sample solution was detected, and the concentration of ochratoxin A in the test sample solution was obtained according to the working curve.

[0070] Figure 2 The operating curve of the electrochemiluminescent ochratoxin A sensing platform obtained in Example 3 is: I = 13289 - 1954 lg c, R 2 =99.8%, where I represents the electrochemiluminescence intensity in au, and c represents the concentration of ochratoxin A in pg / mL; the linear range is 10 pg / mL to 200 ng / mL; LOD=3.23 pg / mL (S / N=3, where S / N represents the ratio of electrochemiluminescence intensity to background noise signal intensity). It can be seen that the working curve obtained based on the electrochemiluminescence ochratoxin A sensing platform of Example 3 has a wider detection range and a lower detection limit, which can achieve more sensitive and accurate trace detection of ochratoxin A.

[0071] The enzyme-free catalytic DNA nanowalker-based electrochemiluminescence ochratoxin A sensing platform designed in this invention can be used for the detection of ochratoxin A in food. This invention introduces the specific recognition of ochratoxin A by DNA aptamers into the field of electrochemiluminescence and designs an enzyme-free catalytic DNA amplification strategy.

[0072] (1) The present invention provides a reliable method for constructing a novel electrochemiluminescent ochratoxin A sensing platform by using DNA aptamers to target and identify ochratoxin A.

[0073] In the field of electrochemiluminescence detection, DNA aptamers demonstrate significant advantages over traditional antigen-antibody detection for the detection of ochratoxin A. DNA aptamers can be synthesized artificially, with a simple, low-cost, and highly consistent preparation process. Their binding specificity to ochratoxin A is high, enabling accurate identification of the target analyte even in complex samples.

[0074] (2) This invention utilizes a signal amplification strategy based on nanomaterials (gold and silver nanomaterials) to enable the DNA aptamer-based electrochemiluminescence sensing platform to achieve ultrasensitive detection with higher sensitivity. Furthermore, the DNA aptamer sensor is easy to operate, suitable for rapid on-site detection, and can be reused multiple times. Its performance remains stable after long-term storage, making it economical and practical.

[0075] This invention synthesizes a material with high electrochemiluminescence properties by loading gold nanoclusters onto silver nanomaterials. Loading gold nanoclusters onto silver nanomaterials effectively suppresses non-radiative diffraction and catalyzes electrochemical reactions. Using the modified gold nanoclusters as luminescent agents in an electrochemiluminescent ochratoxin A sensing platform significantly improves the platform's sensitivity and stability.

[0076] (3) This invention combines magnetic beads with DNA to design an enzyme-free DNA amplification strategy and successfully synthesizes a DNA nanowalker.

[0077] The sensing platform of this invention does not require enzyme catalysis. Enzyme-free sensors are more stable, not limited by the problem of enzyme inactivation, and can operate under a wider range of environmental conditions. Most DNA aptamer sensors require DNase catalysis, and enzymes are easily affected by the environment. Enzyme-free electrochemiluminescence (ECL) sensors have significant advantages over enzyme-catalyzed ECL sensors.

[0078] Magnetic beads are small, magnetic particles, typically composed of a core of magnetic nanomaterials (such as magnetite) coated with a functionalized material (such as polymers or silica). Magnetic beads can act as carriers for DNA, immobilizing DNA probes and enabling the specific capture of target DNA. Their unique physical and chemical properties allow for the rapid separation and enrichment of target DNA under a magnetic field, reducing interference from non-specific binding and thus improving detection sensitivity and specificity. Furthermore, magnetic beads can be combined with signal amplification systems to further enhance ECL signals and improve detection performance. Simultaneously, the high stability of magnetic beads improves the reusability and long-term stability of sensors, giving them significant advantages in biological detection. This invention prepares a DNA nanowalker by linking carboxyl-containing magnetic beads with amino-containing DNA through an amidation reaction. Ochratoxin is added to the double strand formed by the DNA aptamer and the complementary strand, displacing the complementary strand. The complementary strand is then added to the DNA nanowalker, triggering the DNA nanowalker to achieve cyclic amplification. After the magnetic beads are adsorbed and removed, the supernatant is dropped onto the electrode. The DNA strand quencher, carrying a quenching agent, quenches the sensor signal, enabling quantitative detection.

[0079] Experiments have demonstrated that the DNA amplification strategy designed in this invention can effectively identify ochratoxin A and achieve cyclic amplification of the sensor signal. The principle of this invention's detection method is that low concentrations of ochratoxin A can be effectively amplified after passing through a DNA nanowalker, making the detection of even lower concentrations possible. Furthermore, the enzyme-free catalytic DNA nanowalker-based electrochemiluminescent ochratoxin A sensing platform designed in this invention also provides a novel method for the analysis and detection of other toxins with DNA aptamers.

[0080] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. A method for preparing an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker, characterized in that, Includes the following steps: Step 1: Incubate DNA1 and DNA2 to form a double strand. Activate carboxyl-modified magnetic beads with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and incubate with the double strand and N-hydroxysuccinimide to form a windmill-structured nanowalker. Step 2: Polish the glassy carbon electrode with alumina polishing powder; Step 3: Prepare glassy carbon electrodes modified with gold and silver nanomaterials; Step 4: First, mix the ochratoxin A aptamer DNA with DNA3 and incubate for a period of time. Then, add ochratoxin A solution and incubate for a period of time to obtain a mixed solution. Drop the mixed solution onto the DNA nanowalker prepared in Step 1 and incubate for a period of time. Then, add DNA4 and continue incubating for a period of time. Remove the magnetic beads, add the supernatant to the glassy carbon electrode obtained in step 3 and incubate for a period of time. After drying, rinse with PBS buffer solution to obtain an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker. The sequence of DNA1 is shown in SEQ ID No. 1, the sequence of DNA2 is shown in SEQ ID No. 2, the sequence of DNA3 is shown in SEQ ID No. 3, the sequence of DNA4 is shown in SEQ ID No. 4, and the sequence of ochratoxin A aptamer DNA is shown in SEQ ID No.

5.

2. The preparation method according to claim 1, characterized in that, In step one: The 5' end of DNA1 is modified with an amino group; the 5' end of DNA2 is covalently linked to a thiol group via a hexamethylene chain, and the 3' end is modified with the electrochemiluminescent quencher MB.

3. The preparation method according to claim 1, characterized in that, In step three, the preparation method of the glassy carbon electrode modified with gold and silver nanomaterials is as follows: An aqueous solution of gold and silver nanomaterials was drop-coated onto the surface of the glassy carbon electrode obtained in step two, and after drying, a glassy carbon electrode modified with gold and silver nanomaterials was obtained; the gold and silver nanomaterials were silver nanomaterials loaded with gold nanoclusters. The preparation method of the gold and silver nanomaterials is as follows: S1. Add sodium borohydride to silver nitrate solution, place in a water bath at 59-62°C for 5-6 hours, centrifuge and wash with deionized water to obtain silver nanomaterials, disperse the silver nanomaterials in water for later use. S2. Chloroauric acid solution was stirred thoroughly with 4,6-diamino-2-thiopyrimidine for 1 h, left to stand in the dark for 1 day, mixed with silver nanomaterials and citric acid, reacted for 3 days, centrifuged and washed, and collected to obtain gold and silver nanomaterials.

4. The preparation method according to claim 1, characterized in that, The specific operation of step one is as follows: (1) Incubate 5~15 μM, 19 µL~22 µL of DNA1 and 5~15 μM, 19 µL~22 µL of DNA2 at 37 °C for 20 minutes to form double strands; (2) Activate the aqueous solution of 5 mg / mL, 19 ~ 22 µL of carboxyl-modified magnetic beads with 0.1 ~ 0.3 mol / L, 40 µL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for 30 minutes; (3) The activated magnetic beads, 20 µL of double strands and 0.1 ~ 0.3 mol / L, 40 µL of N-hydroxysuccinimide were incubated together for 12 hours to form a windmill-structured nanowalker.

5. The preparation method according to claim 1, characterized in that, The specific operation of step two is as follows: A glassy carbon electrode with a diameter of 4 mm was polished sequentially with alumina polishing powders of 1.0 μm, 0.3 μm, and 0.05 μm, respectively, and then rinsed with ultrapure water to obtain a smooth glassy carbon electrode.

6. The preparation method according to claim 1, characterized in that, The specific operation of step four is as follows: (1) First, mix 10 μM, 19 ~ 22 μL of ochratoxin A aptamer DNA with 10 μM, 19 ~ 22 μL of DNA3 and incubate at 37 °C for 30 minutes; then, add 19 ~ 22 μL of ochratoxin A solution with a concentration of 10 pg / mL to 200 ng / mL and incubate at 37 °C for 30 minutes to obtain a mixed solution; (2) Add the mixed solution to the nanowalker prepared in step one and incubate for 10 minutes, then add 10 μM, 19 ~ 22 μL of DNA4 and continue incubating for 60-80 minutes; (3) Remove the magnetic beads, add the supernatant to the glassy carbon electrode obtained in step 3, dry it, and then rinse it with PBS buffer solution with pH 7.0 ~ 8.2 to obtain an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker.

7. An electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker prepared by the preparation method according to any one of claims 1-6.

8. A method for detecting ochratoxin A based on the sensing platform described in claim 7, characterized in that, The method is as follows: (1) Setup of the electrochemiluminescence system; The test was conducted using a three-electrode system, specifically configured as follows: an Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and an electrochemiluminescent ochratoxin A sensing platform based on an enzyme-free catalytic DNA nanowalker as the working electrode. During the experiment, the electrochemical workstation was used in conjunction with the chemiluminescence detector; (2) The intensity of the electrochemiluminescence signal generated by ochratoxin A standard solution of different concentrations was detected by an electrochemiluminescence system, and the working curve was plotted. (3) The test sample solution is used instead of the ochratoxin A standard solution for determination, and the concentration of ochratoxin A in the test sample solution is obtained according to the working curve.

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