Preparation method of electrochemical sensor for simultaneously detecting aflatoxin B1 and ochratoxin A based on DNA windmill structure

Abstract

This invention presents a method for fabricating an electrochemical sensor based on a DNA windmill structure for the simultaneous detection of AFB1 and OTA. The aptamers of AFB1 and OTA, along with their complementary double-stranded structures Apt-1 / cDNA-1 and Apt-2 / cDNA-2, are respectively connected to magnetic beads. In the presence of AFB1 and OTA, cDNA-1 detaches into the supernatant, while cDNA-2 remains on the magnetic beads. After magnetic separation, PtCuMn / S1 / AQ / HP1 is added to the supernatant, causing DNA windmill blade probe-1 to detach. Pd@Pt / S2 / Thi / HP2 is then added to the precipitate, causing DNA windmill blade probe-2 to detach. The DNA windmill blade probe is then dropped onto an electrode loaded with a DNA windmill backbone to form a DNA windmill structure. Quantitative detection of AFB1 and OTA is achieved by measuring the signal changes of AQ and Thi.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to a method for preparing and applying an electrochemical sensor based on a windmill structure. Background Technology

[0002] Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are two of the most widely distributed and toxic mycotoxins in the food industry, extensively contaminating various foods and animal feeds, including peanuts, rice, and grains. AFB1 is highly hepatotoxic and carcinogenic, and long-term intake can lead to serious diseases such as liver cancer. OTA is characterized by significant nephrotoxicity and also has teratogenic and mutagenic effects; its residues in food can cause irreversible damage to human kidney function. Both toxins often coexist in agricultural products and are chemically stable, making them difficult to remove during food processing through conventional heat treatment or hydrolysis, posing a significant challenge to food safety supervision. Therefore, developing a highly sensitive detection technology that can rapidly, accurately, and simultaneously detect both toxins is of significant practical importance and application value for ensuring food safety and protecting public health.

[0003] Currently, conventional methods for detecting AFB1 and OTA mainly include high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). While these methods offer high sensitivity and accuracy, they are complex, require specialized technicians, have long analysis times, and involve expensive equipment. Compared to traditional methods, electrochemical sensing technology is simpler to operate, has a faster response time, and a shorter analysis cycle. To further improve sensor performance, this method incorporates nucleic acid aptamers (Apt) to specifically recognize the two toxins and achieve simultaneous detection. Hollow nanomaterials PtCuMn and Pd@Pt nanocomposites are introduced to load different signal molecules, increasing electron transfer rates, enriching signal molecules, and enhancing signal response. Simultaneously, a DNA windmill structure is introduced to bind with more signal probes, achieving signal amplification. The electrochemical sensor based on this invention simultaneously detects AFB1 and OTA, exhibiting good stability and high sensitivity, which is beneficial for the application and promotion of this invention. Summary of the Invention

[0004] 1. A method for preparing an electrochemical sensor for simultaneous detection of AFB1 and OTA based on a DNA windmill structure, characterized by comprising the following steps: (1) Preparation of DNA windmill blade probe: First, threonine, PVP, and sodium iodide were dissolved in water and stirred evenly. Chloroplatinic acid, copper nitrate, manganese chloride, and ethanolamine were added, mixed, stirred, and heated. After several washings and drying, hollow structure PtCuMn was obtained. The PtCuMn solution was incubated with DNA strand S1 by oscillation, and then anthraquinone AQ solution, an electrochemical signal molecule, was added for incubation to obtain PtCuMn / S1 / AQ, i.e., DNA windmill blade probe-1. After incubation with hairpin HP1 by oscillation, PtCuMn / S1 / AQ / HP1 was obtained for the detection of AFB1. PVP, ascorbic acid, and potassium bromide were dissolved in water and stirred evenly. After heating and stirring in water until homogeneous, Na2PdCl4 was added and heated and stirred. After several washes, palladium cubes were obtained. Chloroplatinic acid and glucose were dissolved in oleylamine and added to the palladium cubes. After heating and stirring, Pd@Pt with a cubic structure was obtained after washing and drying. The Pd@Pt solution was incubated with DNA strand S2 by shaking to allow DNA strand S2 to be attached to the surface of Pd@Pt. Then, the electrochemical signal molecule Thionium solution was added and incubated by shaking to obtain Pd@Pt / S2 / Thi, i.e., DNA windmill blade probe-2. After incubation with hairpin HP2 by shaking, Pd@Pt / S2 / Thi / HP2 was obtained for the detection of OTA. (2) Construction of an electrochemical sensor for simultaneous detection of AFB1 and OTA: A double-stranded structure Apt-1 / cDNA-1 was obtained by shaking and incubating biotin-labeled AFB1 aptamer Apt-1 and its complementary strand cDNA-1. A double-stranded structure Apt-2 / cDNA-2 was obtained by shaking and incubating OTA aptamer Apt-2 and its complementary strand cDNA-2 labeled with biotin. The two double-stranded structures were connected to magnetic beads via streptavidin-biotin interaction. In the presence of AFB1 and OTA, AFB1 binds to Apt-1, and cDNA-1 detaches into the supernatant. OTA binds to Apt-2, and cDNA-2 remains on the magnetic beads. PtCuMn / S1 / AQ / HP1 was added to the supernatant. DNA-1 opens HP1, causing DNA windmill blade probe-1 to detach. Pd@Pt / S2 / Thi / HP2 is added to the precipitate, and cDNA-2 opens HP2, causing DNA windmill blade probe-2 to detach. DNA strand S3 and DNA strand S4 are oscillated and incubated to form a DNA windmill structure backbone, which is then attached to the electrode. The detached DNA windmill blade probe-1 and DNA windmill blade probe-2 are then dropped onto the electrode surface to form a complete DNA windmill structure with the DNA windmill structure backbone. The electrochemical signals of AQ and Thi are detected using square wave voltammetry. An electrochemical sensor was successfully constructed to achieve quantitative detection of AFB1 and OTA. The introduction of the DNA windmill structure enables signal amplification and simultaneous detection of two toxins, AFB1 and OTA.

[0005] Further specified, in step (1), the concentration of the PtCuMn solution is 1 ~ 5 mg / mL; the concentration ratio of DNA windmill blade probe-1 to hairpin HP1 is 1:1 ~ 3:1; the concentration of Pd@Pt solution is 1 ~ 3 mg / mL; and the concentration ratio of DNA windmill blade probe-2 to hairpin HP2 is 1:1 ~ 3:1.

[0006] Further specified, in step (2), the concentration ratio of DNA chain S3 to DNA chain S4 is 1:1 to 1:2; the concentration ratio of AFB1 aptamer chain Apt-1 to cDNA-1 is 1:1 to 1:2; and the concentration ratio of OTA aptamer chain Apt-2 to cDNA-2 is 1:1 to 1:2.

[0007] Further specifying, in steps (1) and (2), the incubation time is 1 to 10 hours; the incubation temperature is 20 to 50°C; the S1 sequence is 5'-TAT GTG GGC CTA GC-SH-3'; the hairpin HP1 sequence is 5'-GTG CCCTTC GCT AGG CCC ACA TAT GCT AGG CCC ACA TAG AAG GGC ACG AG-3'; the S2 sequence is 5'-GTC CGA TGC TCC-SH-3'; the hairpin HP2 sequence is 5'-TCT TAC AAA GGG AGC ATC GGACAG GAG CAT CGG ACC TTT GTA AGA-3'; and the complementary strand cDNA-1 sequence is 5'-GG GCC TAG CAT ATGTGG GCC TAG CGA AGG GCA CGA GAC ACA GAG AGA CAA CAC GTG CC-3'; the complementary strand cDNA-2 sequence is 5'-GAT GCT CCT GTC CGA TGC TCC CTT TAC GCC ACC CAC ACC CGA TC-Biotin-3'; the S3 sequence is 5'-GCT AGG CCC ACA TAC ATT TTT TTT TTT TTT TAC GCTAGG CCC ACA TA-3'; the S4 sequence is 5'-GGA GCA TCG GAC CCA AAA AAA AAA AAA AACAGG AGC ATC GGA CCC CCC C-SH-3'. Attached Figure Description

[0008] Figure 1This is a schematic diagram illustrating the fabrication method of an electrochemical sensor based on a windmill structure.

[0009] Figure 2 The graphs show the square wave volt-ampere curves of the sensor constructed in Embodiment 1 of the present invention before (dashed line) and after (solid line) the addition of AFB1 and OTA.

[0010] Figure 3 This is the standard curve for the sensor detection of AFB1 and OTA constructed in Embodiment 1 of the present invention.

[0011] Figure 4 This demonstrates the specificity of the sensor constructed in Embodiment 1 of the present invention for AFB1 and OTA in the presence of other interfering substances. Detailed Implementation

[0012] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific examples. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods. Example

[0013] A method for fabricating an electrochemical sensor for simultaneous detection of AFB1 and OTA based on a DNA windmill structure, the implementation method of which is as follows: Figure 1 As shown.

[0014] The specific steps are as follows: (1) Preparation of DNA windmill blade probe: 50 mg threonine, 100 mg PVP, and 30 mg sodium iodide were dissolved in 3.65 mL of water and stirred for 15 min under magnetic stirring. 1 mL of 20 mM chloroplatinic acid, 1 mL of 20 mM copper nitrate, 0.25 mL of 20 mM manganese chloride, and 0.35 mL of ethanolamine were added and mixed. The mixture was heated at 160 °C for 16 h. The product was collected by centrifugation at 10000 rpm for 10 min and dried to obtain the centrally controlled structure PtCuMn. 50 μL of 1.5 mg / mL PtCuMn solution and 50 μL of 20 μM DNA strand S1 treated with reduced disulfide bonds were incubated at 37 °C for 2 h with shaking. After centrifugation, 50 μL of anthraquinone AQ solution was added and incubated at 37 °C with shaking for 2 h to obtain PtCuMn / S1 / AQ, i.e., DNA windmill blade probe-1. This was then mixed with 50 μL of 10 mM chloroplatinic acid, 1 mL of 20 mM copper nitrate, 0.25 mL of 20 mM manganese chloride, and 0.35 mL of ethanolamine. The hairpin HP1 annealed to μM was incubated with shaking at 37℃ for 1 h to obtain PtCuMn / S1 / AQ / HP1 for AFB1 detection; 105 mg PVP, 60 mg ascorbic acid, and 600 mg potassium bromide were dissolved in 8 mL of water, stirred in an oil bath at 80℃ for 10 min, and then 3 mL of 19 mg / mL Na2PdCl4 was added. The mixture was stirred in an oil bath at 80℃ for 3 h, cooled, and centrifuged to collect the product; 1 mL of 1.5 mg palladium cubic solution, 10 mg chloroplatinic acid, and 100 mg glucose were dissolved in oleylamine and stirred in an oil bath at 200℃ for 3 h. After several washes and drying, Pd@Pt with a cubic structure was obtained. 1.5 mg Pd@Pt was dispersed in 1 mL of Tris buffer and incubated with 50 μL of 20 μM DNA strand S2 treated with reducing disulfide bonds at 37℃ for 2 h. h, DNA strand S2 is linked to the Pd@Pt surface, centrifuged, and 50 μL of thionine Thi solution is added and incubated at 37℃ for 2 h with shaking to obtain Pd@Pt / S2 / Thi, i.e., DNA windmill blade probe-2. Then, it is incubated with 50 μL of 10 μM annealed hairpin HP2 with shaking to obtain Pd@Pt / S2 / Thi / HP2 for OTA detection.

[0015] (2) Construction of electrochemical sensors and identification of target substances: First, a magnetic separation system was constructed. 10 μL, 20 μM of biotin-labeled AFB1 aptamer Apt-1 and 10 μL, 20 μM of complementary cDNA-1 were vortexed and incubated to obtain the double-stranded structure Apt-1 / cDNA-1. 10 μL, 20 μM of OTA aptamer Apt-2 and 10 μL, 20 μM of biotin-labeled complementary cDNA-2 were vortexed and incubated to obtain the double-stranded structure Apt-2 / cDNA-2. 5 μL of each was then taken... μL of double-stranded structures Apt-1 / cDNA-1 and Apt-2 / cDNA-2 were linked to magnetic beads via streptavidin and biotin. In the presence of AFB1 and OTA in the magnetic separation system, AFB1 bound to Apt-1, causing cDNA-1 to detach into the supernatant; OTA bound to Apt-2, causing cDNA-2 to detach into the supernatant, while cDNA-2 remained on the magnetic beads. Adding PtCuMn / S1 / AQ / HP1 to the supernatant caused the cDNA-1 in the supernatant to open HP1, resulting in the detachment of DNA windmill probe-1. Adding Pd@Pt / S2 / Thi / HP2 to the precipitate caused the cDNA-2 retained in the precipitate to open HP2, resulting in the detachment of DNA windmill probe-2. After incubating 10 μL of 2 μM DNA strand S3 with 10 μL of 2 μM DNA strand S4 with shaking, the backbone of the DNA windmill structure was formed. 5 μL of the DNA windmill structure was then collected. μL was connected to the electrode, and the detached DNA windmill blade probe-1 and DNA windmill blade probe-2 were dropped onto the electrode surface and combined with the DNA windmill structure backbone to form a complete DNA windmill structure. Finally, square wave voltammetry was used to detect the electrochemical signals of AQ and Thi, realizing the quantitative detection of AFB1 and OTA. The introduction of the DNA windmill structure realizes signal amplification and simultaneous detection of two toxins, AFB1 and OTA.

[0016] (3) Establishment of standard curves: 10 μL of AFB1 and OTA standard solutions of different concentrations were added to step (2). After the reaction, different current signals were obtained. The logarithm of AFB1 and OTA concentrations was used as the abscissa and the current signal was used as the ordinate for linear fitting to establish the standard curves of AFB1 and OTA for the sensor.

[0017] like Figure 2 The figure shows the current detection results of the sensor constructed in Embodiment 1 of the present invention before (dashed line) and after (solid line) the addition of AFB1 and OTA.

[0018] like Figure 3 As shown, this is the standard curve for sensor detection of AFB1 and OTA constructed in Embodiment 1 of the present invention. Example

[0019] A method for fabricating an electrochemical sensor based on a DNA windmill structure for simultaneous detection of AFB1 and OTA, and its practical application, includes the following steps: To verify the specific recognition of AFB1 and OTA by the newly prepared electrochemical biosensor, AFB1 and OTA standards were added to Tris buffer solution to achieve a concentration of 50 ng / mL for AFB1 and OTA in the sample. Standard solutions of six other interfering substances (AFM1, ZEN, FB1, OTB, T-2, and DON) were prepared using Tris buffer solution, each with a concentration of 500 ng / mL. The detection system constructed in Example 1 was used to detect the above six different interfering substance standard solutions and their mixed samples with AFB1 and OTA. The detection results are as follows: Figure 4 As shown, this demonstrates that the method of the present invention exhibits good selectivity for AFB1 and OTA. Example

[0020] A method for fabricating and applying an electrochemical sensor based on a DNA windmill structure for simultaneous detection of AFB1 and OTA, the practical application of which includes the following steps: (1) Food sample processing: For soybean samples, weigh 5 g of soybeans, add 20 mL of methanol-water solution (7:3), vortex to mix, place in a shaker and shake for 20 min, centrifuge at 6000 rpm for 10 min, take the supernatant, filter with a 0.45 μm filter membrane, and use the standard addition method to obtain the spiked sample; For edible oil samples, weigh 5 g of edible oil, add 20 mL of petroleum ether, shake to dissolve the oil sample, add 10 mL of methanol-water solution (7:3), vortex to mix, let stand to separate the layers, take the lower layer extract and put it in a refrigerator, freeze at -15℃ for 20 min, filter the extract with a 0.45 μm filter membrane, and use the standard addition method to obtain the spiked sample.

[0021] (2) Sample detection: The electrical signal was measured according to the steps in Example 1, and the concentrations of AFB1 and OTA in the sample were obtained by substituting it into the standard curve.

[0022] (3) When using soybeans and edible oil as actual samples for determination, different concentrations of AFB1 and OTA standards were added to the samples respectively. Take 10 μL of sample solution and measure the current signal according to steps (1)-(2) of Example 1. Substitute the signal into the standard curve detected in Example 1 to obtain the concentrations of AFB1 and OTA in the sample. Each sample was measured three times and the average value was taken. The average recovery rate of the sensor was calculated to be 95.8% ~ 102.6%.

[0023] The novel electrochemical sensor demonstrated by the fabricated material exhibits high sensitivity, good selectivity, simultaneous detection, and good stability for the detection of AFB1 and OTA. Testing on actual samples shows that the fabricated sensor has significant practical application value.

[0024] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention and do not limit the invention in any way. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications should fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for fabricating an electrochemical sensor based on a DNA windmill structure for simultaneous detection of AFB1 and OTA, characterized in that, Includes the following steps: (1) Preparation of DNA windmill blade probe: First, threonine, PVP, and sodium iodide were dissolved in water and stirred evenly. Chloroplatinic acid, copper nitrate, manganese chloride, and ethanolamine were added, mixed, stirred, and heated. After several washings and drying, hollow structure PtCuMn was obtained. The PtCuMn solution was incubated with DNA strand S1 by oscillation, and then anthraquinone AQ solution, an electrochemical signal molecule, was added for incubation to obtain PtCuMn / S1 / AQ, i.e., DNA windmill blade probe-1. After incubation with hairpin HP1 by oscillation, PtCuMn / S1 / AQ / HP1 was obtained for the detection of AFB1. PVP, ascorbic acid, and potassium bromide were dissolved in water and stirred evenly. After heating and stirring in water until homogeneous, Na2PdCl4 was added and heated and stirred. After several washes, palladium cubes were obtained. Chloroplatinic acid and glucose were dissolved in oleylamine and added to the palladium cubes. After heating and stirring, Pd@Pt with a cubic structure was obtained after washing and drying. The Pd@Pt solution was incubated with DNA strand S2 by shaking to allow DNA strand S2 to be attached to the surface of Pd@Pt. Then, the electrochemical signal molecule Thionium solution was added and incubated by shaking to obtain Pd@Pt / S2 / Thi, i.e., DNA windmill blade probe-2. After incubation with hairpin HP2 by shaking, Pd@Pt / S2 / Thi / HP2 was obtained for the detection of OTA. (2) Construction of an electrochemical sensor for simultaneous detection of AFB1 and OTA: A double-stranded structure Apt-1 / cDNA-1 was obtained by shaking and incubating biotin-labeled AFB1 aptamer Apt-1 and its complementary strand cDNA-1. A double-stranded structure Apt-2 / cDNA-2 was obtained by shaking and incubating OTA aptamer Apt-2 and its complementary strand cDNA-2 labeled with biotin. The two double-stranded structures were connected to magnetic beads via streptavidin-biotin interaction. In the presence of AFB1 and OTA, AFB1 binds to Apt-1, and cDNA-1 detaches into the supernatant. OTA binds to Apt-2, and cDNA-2 remains on the magnetic beads. PtCuMn / S1 / AQ / HP1 was added to the supernatant. DNA-1 opens HP1, causing DNA windmill blade probe-1 to detach. Pd@Pt / S2 / Thi / HP2 is added to the precipitate, and cDNA-2 opens HP2, causing DNA windmill blade probe-2 to detach. DNA strand S3 and DNA strand S4 are oscillated and incubated to form a DNA windmill structure backbone, which is then attached to the electrode. The detached DNA windmill blade probe-1 and DNA windmill blade probe-2 are then dropped onto the electrode surface to form a complete DNA windmill structure with the DNA windmill structure backbone. The electrochemical signals of AQ and Thi are detected using square wave voltammetry. An electrochemical sensor was successfully constructed to achieve quantitative detection of AFB1 and OTA. The introduction of the DNA windmill structure enables signal amplification and simultaneous detection of two toxins, AFB1 and OTA.

2. The method for preparing an electrochemical sensor for simultaneous detection of AFB1 and OTA based on a DNA windmill structure according to claim 1, characterized in that, In step (1), the concentration of the PtCuMn solution is 1 ~ 5 mg / mL; the concentration ratio of DNA windmill blade probe-1 to hairpin HP1 is 1:1 ~ 3:1; the concentration of Pd@Pt solution is 1 ~ 3 mg / mL; and the concentration ratio of DNA windmill blade probe-2 to hairpin HP2 is 1:1 ~ 3:

1.

3. The method for fabricating an electrochemical sensor based on a windmill structure according to claim 1, characterized in that, In step (2), the concentration ratio of DNA chain S3 to DNA chain S4 is 1:1 to 1:2; the concentration ratio of AFB1 aptamer chain Apt-1 to cDNA-1 is 1:1 to 1:2; and the concentration ratio of OTA aptamer chain Apt-2 to cDNA-2 is 1:1 to 1:

2.

4. The method for fabricating an electrochemical sensor based on a windmill structure according to claim 1, characterized in that, In steps (1) and (2), the incubation time is 1 to 10 hours and the incubation temperature is 20 to 50°C.

5. The method for fabricating an electrochemical sensor based on a windmill structure according to claim 1, characterized in that, In steps (1) and (2), the S1 sequence is 5'-TAT GTG GGC CTA GC-SH-3'; the hairpin HP1 sequence is 5'-GTG CCC TTC GCT AGG CCC ACA TAT GCT AGG CCC ACA TAG AAG GGC ACG AG-3'; the S2 sequence is 5'-GTC CGA TGC TCC-SH-3'; the hairpin HP2 sequence is 5'-TCT TAC AAAGGG AGC ATC GGA CAG GAG CAT CGG ACC TTT GTA AGA-3'; the complementary strand cDNA-1 sequence is 5'-GGGCC TAG CAT ATG TGG GCC TAG CGA AGG GCA CGA GAC ACA GAG AGA CAA CAC GTG CC-3'; and the complementary strand cDNA-2 sequence is 5'-GAT GCT CCT GTC CGA TGC TCC CTT TAC GCC ACC CACACC. CGA TC-Biotin-3'; the S3 sequence is 5'-GCT AGG CCC ACA TAC ATT TTT TTT TTT TTTTAC GCT AGG CCC ACA TA-3'; the S4 sequence is 5'-GGA GCA TCG GAC CCA AAA AAA AAAAAA AAC AGG AGC ATC GGA CCC CCC C-SH-3'.

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