Method for detecting aflatoxin B1 based on ratio electrochemical aptamer sensor
Through ratio electrochemical aptamer sensor, Zr-MOF is prepared by solvothermal reaction and combined with catalytic hairpin assembly strategy, an electrochemical aptamer sensor is constructed, which solves the problem of low detection sensitivity of aflatoxin B1 in the prior art and achieves high sensitivity AFB1 detection.
Patent Information
- Application Number
- CN202510648024.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-20
- Publication Date
- 2025-08-19
AI Technical Summary
The prior art is difficult to detect aflatoxin B1 with high sensitivity, and traditional methods are difficult to completely eliminate its toxicity.
A ratio electrochemical aptamer sensor was used to prepare Zr-MOF through solvothermal reaction, and combined with the catalytic hairpin assembly strategy, an electrochemical aptamer sensor was constructed, and the peak current ratio ΔIFc/ΔIMOF of the Zr-MOF signal and the ferrocene signal was used to achieve detection.
The detection sensitivity of aflatoxin B1 is improved, and the rapid and simple AFB1 detection is achieved, which can accurately reflect the actual concentration of AFB1.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of electrochemical technology, and in particular to a method for detecting aflatoxin B1 based on a ratiometric electrochemical aptamer sensor. Background Art
[0002] Aflatoxins are secondary metabolites produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. Their structures contain a difuran ring and coumarin. They are highly physically and chemically stable, with an optimal temperature for toxin production between 26°C and 28°C. They are extremely stable and difficult to decompose, with decomposition temperatures exceeding 270°C. Conventional cooking methods are incapable of completely eliminating their toxicity. Currently, there are approximately 20 AFT derivatives, primarily including aflatoxin B1 (AFB1), AFB2, AFM1, AFM2, AFG1, and AFG2. Among these derivatives, AFB1 is the most widespread, most toxic, and most harmful, making it one of the most toxic mycotoxins.
[0003] AFB1 is widely distributed in nature and exists in animals, plants, various nuts and soil, especially in moldy peanuts and walnuts. AFB1 can inhibit protein synthesis. Its toxicity is 68 times that of arsenic and 10 times that of potassium cyanide. For the human body, long-term intake of this substance may cause liver damage, and eventually lead to diseases such as liver cancer and gastric cancer. Long-term intake of small amounts can cause chronic diseases, such as growth retardation in children and fibrotic lesions. Therefore, it poses a global threat to public health. For the sake of human health and environmental safety, many countries have determined the maximum residual level of AFB1 to be 2-20μg kg -1 within the range.
[0004] In recent years, electrochemical biosensors have been widely used in fields such as biocontamination and environmental monitoring due to their fast response, high sensitivity, and portability. To significantly enhance sensor sensitivity, researchers often incorporate ratiometric strategies and nucleic acid signal amplification techniques to construct highly sensitive sensing interfaces, enabling highly sensitive detection of target molecules. Summary of the Invention
[0005] The purpose of the present invention is to improve the sensitivity of AFB1 detection and to construct a fast and simple electrochemical aptamer sensor for detecting AFB1 by combining a ratiometric strategy with a catalytic hairpin assembly strategy.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] A method for detecting aflatoxin B1 based on a ratiometric electrochemical aptamer sensor, specifically comprising:
[0008] S1. Dissolve zirconium salt and diaminoterephthalic acid (BDC-NH2) in N,N-dimethylformamide (DMF), add acetic acid (HAc), and then transfer to a high-pressure reactor. React at 80-140°C for 12-48 hours, centrifuge, wash, and dry to obtain UiO-66-NH2 (Zr-MOF) particles;
[0009] S2, H Anchor Add to Zr-MOF solution, incubate at 37°C, collect the precipitate by centrifugation, and then dissolve the precipitate in ultrapure water and resuspend it to obtain Zr-MOF / H Anchor Complex, H Anchor It is a hairpin DNA with the 5' end modified by a phosphate group;
[0010] S3, containing C Apt The solution was incubated with the target AFB1 at 37°C. Apt Can specifically bind to AFB1 to form C Apt / AFB1, then H1 and H2 are added to the solution, the hairpin structure H1 opens and connects to C Apt / AFB1 combined to form a H1-CApt / AFB1 double-stranded structure, which reacted at 37°C to obtain the output DNA. H1 was a hairpin DNA modified with ferrocene (Fc) at its 3' end, and H2 was a hairpin DNA. Anchor Partial base pairing, H1 and H2 partially base pairing;
[0011] S4. Subsequently, the cultured Zr-MOF / H Anchor The complex was coated on the mirror-like surface of glassy carbon electrode (GCE) to obtain H Anchor / Zr-MOF / GCE, and then drop the output DNA onto the electrode and incubate at 37°C to obtain CHA / H Anchor / Zr-MOF / GCE electrode;
[0012] S5, using a three-electrode system, the working electrode is CHA / H Anchor / Zr-MOF / GCE electrode, platinum electrode (Pt) was selected as the counter electrode, saturated calomel electrode (SCE) was used as the reference electrode, and electrochemical tests were carried out in PBS buffer solution. The peak current ΔI Fc and ΔI MOF , calculate ΔI Fc / ΔI MOF , ΔI Fc / ΔIMOF Substitute the corresponding standard curve to obtain the actual concentration of AFB1.
[0013] In step S5 of the present invention, an AC voltammetry test is performed in 0.1 M phosphate buffer (PBS, pH=7.0) with a potential of 4 mV, a frequency of 100 Hz, and an amplitude of 25 mV.
[0014] In step S1 of the present invention, the mass ratio of the zirconium salt to the organic ligand is 1.285:1.
[0015] The zirconium salt in the present invention is ZrCl4.
[0016] Step S1 is a solvent thermal reaction at 80-140°C for 12-48 hours, wherein the reaction temperature is preferably 90-130°C, more preferably 120°C.
[0017] In step S2 of the present invention, Zr-MOF particles are dispersed in ultrapure water containing ethanol and a perfluorosulfonic acid type polymer solution Nafion, and a Zr-MOF solution is obtained by ultrasound. The mass concentration of Zr-MOF particles in the Zr-MOF solution is 1 mg / mL, and the volume ratio of ultrapure water, ethanol and the perfluorosulfonic acid type polymer solution Nafion in the Zr-MOF solution is 100:40:1.
[0018] In step S3 of the present invention, the molar ratio of H1 to H2 is 1.4:1, C Apt The molar ratio with H2 is 1:1.
[0019] In step S4, the glassy carbon electrode is polished with aluminum oxide powder to obtain a mirror-like surface, and then ultrasonically treated in ethanol and ultrapure water, and finally washed with water and dried for use.
[0020] Compared with the prior art, the present invention has the following advantages: (1) Zr-MOF with controllable morphology is prepared by solvent thermal reaction; (2) the constructed sensor can simultaneously detect two electrochemical signals, namely Zr-MOF signal and ferrocene (Fc) signal, by comparing the peak current ratio ΔI of the two electrochemical signals. Fc / ΔI MOF To achieve accurate detection of aflatoxin B1, the ratio detection method has higher detection sensitivity than single electrochemical signal detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Schematic diagram of the fluorescence of the CHA strategy using the fluorescent reporter probe FQ.
[0022] Figure 2 is a graph of CHA fluorescence response at 37°C.
[0023] Figure 3This is the XRD pattern of Zr-MOF.
[0024] Figure 4 This is the TEM image of Zr-MOF.
[0025] Figure 5 This is the ACV response diagram when the Zr-MOF solution is directly drop-coated on the GCE.
[0026] Figure 6 This is the peak current diagram when Zr-MOF is modified on different electrodes.
[0027] Figure 7 It is Zr-MOF / H Anchor High-resolution O1s spectrum.
[0028] Figure 8 In 5 mM [Fe(CN)6] 3- / 4- EIS tests were carried out in the solution, namely GCE, H Anchor / Zr-MOF / GCE and CHA / H Anchor EIS diagram of / Zr-MOF / GCE.
[0029] Figure 9 Figure 2 is a graph of ACV responses in the presence and absence of 0.2 ng / mL AFB1. DETAILED DESCRIPTION
[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0031] Unless otherwise stated, when describing numerical ranges herein, the endpoint values are included. When two or more preferred or exemplary numerical values or numerical ranges are given, it goes without saying that all ranges formed by combining different numerical values or endpoints are also included in the scope of the present invention.
[0032] These and other aspects, features and advantages of the present invention will become apparent to those skilled in the art through the detailed description below. In this context, any feature or any embodiment from one aspect of the present invention may be used in any other aspect of the present invention. Furthermore, it is self-evident that the embodiments herein included are intended to describe and illustrate the present invention, not to limit the present invention, and in particular, the present invention is not limited to these embodiments.
[0033] An exemplary embodiment of a method for detecting aflatoxin B1 based on a ratiometric electrochemical aptasensor according to the present invention will be described below. The embodiment is merely illustrative and the present invention is not limited thereto.
[0034] Specifically, the following steps are included:
[0035] S1. Dissolve zirconium salt and diaminoterephthalic acid (BDC-NH2) in N,N-dimethylformamide (DMF), add acetic acid (HAc), and then transfer to a high-pressure reactor. React at 80-140°C for 12-48 hours, centrifuge, wash, and dry to obtain UiO-66-NH2 (Zr-MOF) particles;
[0036] S2, H Anchor Add to Zr-MOF solution, incubate at 37°C, collect the precipitate by centrifugation, and then dissolve the precipitate in ultrapure water and resuspend it to obtain Zr-MOF / H Anchor Complex, H Anchor It is a hairpin DNA with the 5' end modified by a phosphate group;
[0037] S3, will contain C Apt The solution was incubated with the target AFB1 at 37°C. Apt Can specifically bind to AFB1 to form C Apt / AFB1, then H1 and H2 are added to the solution, the hairpin structure H1 opens and connects to C Apt / AFB1 combined to form a H1-CApt / AFB1 double-stranded structure, and reacted at 37°C to obtain the output DNA, H1 is a hairpin DNA modified with ferrocene Fc at the 3' end, H2 is a hairpin DNA, H1 and H Anchor Partial base pairing, H1 and H2 partially base pairing;
[0038] S4. Subsequently, the cultured Zr-MOF / H Anchor The complex was coated on the mirror-like surface of glassy carbon electrode (GCE) to obtain H Anchor / Zr-MOF / GCE, and then drop the output DNA onto the electrode and incubate at 37°C to obtain CHA / H Anchor / Zr-MOF / GCE;
[0039] S5, using a three-electrode system, CHA / H Anchor / Zr-MOF / GCE was used as the working electrode and electrochemical tests were carried out in PBS buffer solution. The peak current ΔI Fc and ΔI MOF , calculate ΔI Fc / ΔI MOF , ΔI Fc / ΔIMOF Substitute the corresponding standard curve to obtain the actual concentration of AFB1.
[0040] Electrochemical impedance spectrum (EIS) characterization was carried out in the presence of 0.1MKCl5mmol / L[Fe(CN)6] 3- / 4- AC voltammetry (ACV) was performed in 0.1 M phosphate buffer (PBS, pH = 7.0) at a potential of 4 mV, a frequency of 100 Hz, and an amplitude of 25 mV.
[0041] In step S1 of the present invention, the mass ratio of the zirconium salt to the organic ligand is 1.285:1.
[0042] The zirconium salt in the present invention is ZrCl4.
[0043] Step S1 is a solvothermal reaction at 80-140° C. for 12-48 hours. The reaction temperature is preferably 90-130° C., more preferably 120° C., for example, 80° C., 90° C., 100° C., 110° C., 120° C., 130° C., 140° C., or a range defined by any two of the above values.
[0044] In step S2 of the present invention, Zr-MOF particles are dispersed in ultrapure water containing ethanol and a perfluorosulfonic acid type polymer solution Nafion, and a Zr-MOF solution is obtained by ultrasound. The mass concentration of Zr-MOF particles in the Zr-MOF solution is 1 mg / mL, and the volume ratio of ultrapure water, ethanol and the perfluorosulfonic acid type polymer solution Nafion in the Zr-MOF solution is 100:40:1.
[0045] In step S2 of the present invention, the phosphate-modified DNA (H Anchor ) are connected to Zr-MOF particles through Zr-OP bonds to form Zr-MOF / H Anchor The composite was drop-coated on a glassy carbon electrode (GCE) to generate an electrochemical signal of Zr-MOF.
[0046] In step S3 of the present invention, the molar ratio of H1 to H2 is 1.4:1, C Apt The molar ratio of C to target AFB1 is 5:7. Apt The molar ratio with H2 is 1:1.
[0047] In step S4, the glassy carbon electrode is polished with aluminum oxide powder to obtain a mirror-like surface, and then ultrasonically treated in ethanol and ultrapure water, and finally washed with water and dried for use.
[0048] The following further illustrates a method for constructing a ratiometric electrochemical aptasensor and its application in the detection of aflatoxin B1 through specific embodiments.
[0049] Example 1
[0050] C Apt The mixture of H1, H2 and FQ was annealed at 95℃ for 5 minutes and then cooled to 25℃ at a rate of 0.1℃ / s before use. CHA fluorescence kinetics was tested in 1×TNaK buffer. The final standard reaction mixture of CHA contained 100nM C Apt , 100 nM H1, 400 nM H2, and 100 nM FQ reporter duplex, with and without AFB1. In the reporter duplex, [F] is 100 nM, half the concentration of [Q]. Fluorescence signals from 40 μL of the CHA mixture were recorded every 1 min at 37°C on a LineGene Mini S real-time quantitative polymerase chain reaction analyzer.
[0051] All oligonucleotide sequences used in the present invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The specific sequences are shown in the table below.
[0052]
[0053]
[0054] like Figure 1 As shown, the fluorescence schematic diagram of the CHA strategy is to introduce the fluorescent reporter probe FQ: in the presence of the target AFB1, C Apt Can specifically bind to AFB1 to form C Apt / AFB1, and then open the Fc-tagged hairpin structure H1 to form H1-CApt / AFB1 double-stranded structure. In the presence of another hairpin structure H2, because H1 and H2 form a more stable double-stranded structure, C Apt / AFB1 is displaced to form H1-H2 hybrid. The released C Apt AFB1 can trigger another cycle that facilitates signal amplification and error correction. Subsequently, the product H1-H2 binds to FQ, causing Q to detach from FQ and generate fluorescence.
[0055] Figure 2 This is the CHA fluorescence response diagram at 37°C. It can be seen from the figure that the fluorescence reaction rate is significantly enhanced in the presence of target AFB1, proving the feasibility of the CHA process.
[0056] Example 2
[0057] (1) Preparation of Zr-MOF: 0.0315 g zirconium tetrachloride (ZrCl4) and 0.0245 g diaminoterephthalic acid (BDC-NH2) were dissolved in 26.4 mL N,N-dimethylformamide (DMF); then, 3.6 mL acetic acid (HAc) was added to the solution and ultrasonicated for 25 min; the mixture was then transferred to a stainless steel autoclave lined with polytetrafluoroethylene and heated at 120 °C for 24 h to obtain Zr-MOF; the product was then cooled to room temperature, centrifuged, and washed six times with a mixture of methanol:DMF (volume ratio 1:4) at 8000 rpm for 6 min. The final precipitate was dried in a vacuum drying oven at 90 °C overnight to obtain Zr-MOF particles.
[0058] (2) Preparation of Zr-MOF / HAnchor: The prepared Zr-MOF particles (1 mg) were dispersed in 1 mL of ultrapure water containing 400 μL of ethanol and 10 μL of Nafion, and the Zr-MOF solution was obtained by ultrasonication for 20 min. 60 μL of the solution was then mixed with 20 μL of 2 μMH Anchor The mixture was mixed, incubated at 37°C for 10 h, and centrifuged three times to collect the precipitate. The precipitate was then dissolved in ultrapure water, resuspended, and stored at 4°C for later use.
[0059] (3) Synthesis of output DNA: 0.5 μM of target AFB1 was incubated with different concentrations at 37°C for 6 h to form a primer chain (C Apt / AFB1); subsequently, 0.7 μM H1 and 0.5 μM H2 were added to the CApt / AFB1 complex and reacted at 37°C for 2 h to obtain the output DNA (all DNA concentrations were final concentrations).
[0060] (4) Construction of ratiometric electrochemical aptasensor: A glassy carbon electrode (GCE, Φ = 3 mm) was polished with aluminum oxide powder (0.3 μM and 0.05 μM) to obtain a mirror-like surface, which was then ultrasonicated in ethanol and ultrapure water for 40 s and finally washed with water and dried for use. Subsequently, the cultured Zr-MOF / H Anchor The composite was coated on the mirror-like surface of GCE to obtain H Anchor / Zr-MOF / GCE; Then, 6 μL of output DNA was dropped onto the electrode and incubated at 37 °C to obtain CHA / H Anchor / Zr-MOF / GCE electrode was rinsed with phosphate buffer before electrochemical testing.
[0061] Electrochemical characterization and testing of biosensors
[0062] (1) Preparation of PBS buffer: First, accurately weigh potassium dihydrogen phosphate (1.3612 g) and potassium chloride (0.7455 g), add deionized water, stir and mix them until completely dissolved, and then adjust the volume to prepare 100 mL of acidic solution. In addition, accurately weigh potassium chloride (0.7455 g) and dipotassium hydrogen phosphate (1.7418 g), add deionized water, stir and mix them until completely dissolved, and then adjust the volume to prepare 100 mL of alkaline solution. Finally, the prepared acidic solution and alkaline solution are mixed and adjusted to a pH of 7.00. The resulting solution is PBS buffer.
[0063] (2) Potassium ferrocyanide solution ([Fe(CN)6] 3- / 4- Preparation: First, accurately weigh 0.1646 g of potassium ferrocyanide, 0.7455 g of potassium chloride, and 0.2112 g of potassium ferrocyanide. Mix these in a beaker, add deionized water, and stir to dissolve rapidly. The resulting solution will be a yellow-green color. Next, quickly transfer the resulting solution to a 100 mL brown volumetric flask, add deionized water to the mark, securely stopper the flask, invert and shake to mix thoroughly, then quickly transfer to a brown bottle and let stand to obtain a 5 mM potassium ferrocyanide solution.
[0064] (3) Electrochemical measurement: A three-electrode system is used, that is, the working electrode is a glassy carbon electrode (GCE), H Anchor / Zr-MOF / GCE or CHA / H Anchor / Zr-MOF / GCE, platinum electrode (Pt) was selected as the counter electrode, and silver / silver chloride (Ag / AgCl) electrode was selected as the reference electrode, and AC impedance spectroscopy and AC voltammetry tests were performed.
[0065] Electrochemical impedance spectrum (EIS) characterization was performed at 5 mmol / L [Fe(CN)6] 3- / 4- The measurements were performed in a 0.1 M KCl solution with a frequency range of 100 kHz to 0.1 Hz and an amplitude of 5.0 mV.
[0066] AC voltammetry (ACV) was monitored in 0.1 M phosphate buffer (PBS, pH = 7.0) with a potential of 4 mV, a frequency of 100 Hz, and an amplitude of 25 mV.
[0067] Figure 3 The XRD pattern of Zr-MOF was compared with the standard card to confirm the peak position, proving the successful preparation of Zr-MOF.
[0068] Figure 4This is the TEM image of Zr-MOF. It can be seen from the figure that Zr-MOF presents a good regular octahedral structure, which proves the successful preparation of Zr-MOF.
[0069] Figure 5 The ACV response diagram of Zr-MOF / GCE of the same electrode is shown in Figure 2. Zr-MOF solution is directly drop-coated on GCE to obtain Zr-MOF / GCE. Figure 5 It can be seen that Zr-MOF has an obvious oxidation peak current at 0.8V, and the peak current tends to be stable in different tests.
[0070] Figure 6 This is the peak current diagram of AVC test after Zr-MOF is modified to different electrodes. Figure 6 It can be seen that Zr-MOF has good reproducibility.
[0071] from Figure 7 It can be seen that Zr-MOF and H Ancnor The connection is through Zr-OP bond.
[0072] Figure 8 It is GCE, H Anchor / Zr-MOF / GCE and CHA / H Anchor / Zr-MOF / GCE were used as working electrodes to conduct EIS test results. Anchor The Rct of GCE modified with Zr-MOF is greater than that of bare GCE, which may be due to the interaction between negatively charged DNA molecules and negatively charged [Fe(CN)6] 3- / 4- As expected, CHA / H Anchor When the / Zr-MOF / GCE was used as the working electrode, the Rct increased significantly again after the CHA strategy was triggered in the presence of AFB1. These results indicate the successful construction of the electrochemical aptamer sensor platform.
[0073] Figure 9 Figure 1 shows the ACV response graph for the presence and absence of AFB1. The graphs with AFB1 of Fc and without AFB1 of Fc show the current changes for Fc in the presence and absence of AFB1, respectively. The graphs with AFB1 of MOF and without AFB1 of MOF show the current changes for MOF in the presence and absence of AFB1, respectively. Both Fc and MOF exhibited significant electrochemical responses. However, in the absence of target AFB1, the peak current for Fc decreased significantly, while that for Zr-MOF remained almost unchanged. These results demonstrate the feasibility of ratiometric electrochemical aptasensor for detecting AFB1.
[0074] The present invention first prepares Zr-MOF by solvent thermal reaction, the purpose of which is to connect the subsequent DNA and serve as an electroactive material. Anchor ) to obtain Zr-MOF / H Anchor , which was then drop-coated on GCE to generate the electrochemical signal ΔI of Zr-MOF MOF Since C Apt The number of neutral base locks has a certain influence on the reaction rate of CHA. Too many locks will reduce the reaction rate, while too few locks will lead to high background signal. Apt The number of toehold locks in the target AFB1 was optimized. Apt Can specifically bind to AFB1 to form C Apt / AFB1, then opens the Fc-tagged hairpin structure H1 to form H1-C Apt / AFB1 double-stranded structure. In the presence of another hairpin structure H2, H1 and H2 form a more stable double-stranded structure, which makes C Apt / AFB1 is displaced to form H1-H2 hybrid. The released C Apt / AFB1 can trigger another cycle, which is beneficial for signal amplification and error correction. Anchor Base pairing, product H1-H2 and Zr-MOF / H on the electrode Anchor Binding brings Fc close to the electrode surface, generating a strong electrochemical signal ΔI Fc ΔI Fc / ΔI MOF (ΔI Fc The electrochemical signal is enhanced and ΔI MOF The peak current ratio of the electrochemical signal (without the change of electrochemical signal) can accurately reflect the actual concentration of AFB1, and finally achieve the sensitive detection of AFB1.
Claims
1. A method for detecting aflatoxin B1 based on a ratiometric electrochemical aptamer sensor, characterized in that: Specifically: S1. Dissolve zirconium salt and diaminoterephthalic acid in N,N-dimethylformamide, add acetic acid, and then transfer to a high-pressure reactor. React at 80-140° C. for 12-48 hours, centrifuge, wash, and dry to obtain Zr-MOF particles. S2, H Anchor Add to Zr-MOF solution, incubate at 37°C, collect the precipitate by centrifugation, and then dissolve the precipitate in ultrapure water and resuspend it to obtain Zr-MOF / H Anchor Complex, H Anchor It is a hairpin DNA with the 5' end modified by a phosphate group; S3, will contain C Apt The solution was incubated with the target AFB1 at 37°C. Apt Can specifically bind to AFB1 to form C Apt / AFB1, then H1 and H2 are added to the solution, the hairpin structure H1 opens and connects to C Apt / AFB1 combined to form a H1-CApt / AFB1 double-stranded structure, which reacted at 37°C to obtain the output DNA. H1 was a hairpin DNA modified with ferrocene at its 3' end, H2 was a hairpin DNA, and H1 and H Anchor Partial base pairing, H1 and H2 partially base pairing; S4. Subsequently, the cultured Zr-MOF / H Anchor The complex was coated on the mirror-like surface of glassy carbon electrode (GCE) to obtain H Anchor / Zr-MOF / GCE, and then drop the output DNA onto the electrode and incubate at 37°C to obtain CHA / H Anchor / Zr-MOF / GCE electrode; S5, using a three-electrode system, that is, the working electrode is CHA / H Anchor / Zr-MOF / GCE electrode, platinum electrode was selected as counter electrode, saturated calomel electrode was used as reference electrode, and electrochemical test was carried out in PBS buffer solution. The peak current ΔI Fc and ΔI MOF , calculate ΔI Fc / ΔI MOF , ΔI Fc / ΔI MOF Substitute the corresponding standard curve to obtain the actual concentration of AFB1.
2. The method for detecting aflatoxin B1 based on a ratiometric electrochemical aptasensor according to claim 1, characterized in that: H Anchor The sequence is 5'-CGAGTGCTGCGTAGTGACAAGGCAACTGCAATTCACTACG-3', C Apt The sequence is 5'-AATCAGCGCTGACACGTGTTGTCTCTCTGTGTCTCGTGTCAGCGCTGATTGCAGTTGCAAATTGGCT-3', the H1 sequence is 5'-AGCCAATTTGCAACTGCAATCAGCGCTGACCATCCTGCTAGCATCAGCGCTGATTGCAGTTGCCTTGTCACTACGCAGCAC-3', and the H2 sequence is 5'-CAGCGCTGATGCTAGCAGGATGGTCAGCGCTGATTGCAGTTGCCATCCTGCTAGCA-3'.
3. The method for detecting aflatoxin B1 based on a ratiometric electrochemical aptasensor according to claim 1, characterized in that: In step S5, an AC voltammetry test is performed in 0.1 M phosphate buffer (PBS, pH=7.0) with a potential of 4 mV, a frequency of 100 Hz, and an amplitude of 25 mV.
4. The method for detecting aflatoxin B1 based on a ratiometric electrochemical aptasensor according to claim 1, characterized in that: In step S1, the mass ratio of the zirconium salt to the organic ligand is 1.285:
1.
5. The method for detecting aflatoxin B1 based on a ratiometric electrochemical aptasensor according to claim 1, characterized in that: The zirconium salt is ZrCl4.
6. The method for detecting aflatoxin B1 based on a ratiometric electrochemical aptasensor according to claim 1, characterized in that: Step S1 is to carry out a solvothermal reaction at 80-140° C. for 12-48 hours.
7. The method for detecting aflatoxin B1 based on a ratiometric electrochemical aptasensor according to claim 1, characterized in that: In step S2, Zr-MOF particles are dispersed in ultrapure water containing ethanol and a perfluorosulfonic acid type polymer solution Nafion, and a Zr-MOF solution is obtained by ultrasonication. The mass concentration of Zr-MOF particles in the Zr-MOF solution is 1 mg / mL, and the volume ratio of ultrapure water, ethanol, and a perfluorosulfonic acid type polymer solution Nafion in the Zr-MOF solution is 100:40:
1.
8. The method for detecting aflatoxin B1 based on a ratiometric electrochemical aptasensor according to claim 1, characterized in that: In step S3, the molar ratio of H1 to H2 is 1.4:1, C Apt The molar ratio with H2 is 1:
1.
9. The method for detecting aflatoxin B1 based on a ratiometric electrochemical aptasensor according to claim 1, characterized in that: In step S4, the glassy carbon electrode is polished with aluminum oxide powder to obtain a mirror-like surface, and then ultrasonically treated in ethanol and ultrapure water, and finally washed with water and dried for use.