Self-powered biosensor for detecting aflatoxin B1, application and detection method
Patent Information
- Application Number
- CN202610959730.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-30
- Publication Date
- 2026-10-20
AI Technical Summary
从而克服现有检测黄曲霉素B1准确率低、灵敏度低、携带不便和操作复杂等的缺点
[0035]与现有技术相比，本发明的有益效果：本发明通过Au@HPC复合材料显著提升了电极导电性与酶负载效率，结合核酸外切酶介导的信号放大策略，检测限低至1.52 fg/mL，线性范围覆盖0.01～104 pg/mL，灵敏度显著优于现有同类检测方法；实际样品检测中加标回收率达87.9%～102.8%，与液相色谱法结果高度吻合，具备优异的准确度和可靠性。在材料设计上，本发明采用分级多孔碳（HPC）作为基底材料，其内部贯通的微孔、介孔和大孔结构能够为葡萄糖氧化酶和胆红素氧化酶提供充足的负载空间，同时大孔通道有利于底物和产物的快速扩散传质；金纳米粒子的原位修饰不仅显著降低了电极的电荷转移阻抗，还通过Au-S键和静电吸附作用增强了适配体与cDNA的牢固固定，从而大幅提高了生物识别界面的稳定性和信号转换效率。这种材料结构上的双重优势，确保了酶生物燃料电池能够输出更高且更稳定的开路电压和瞬时电流，为后续超灵敏检测奠定了坚实的能量基础。同时，基于酶生物燃料电池构建的自供能传感器无需外部电源、结构简单、易于小型化，尤其适用于食品安全现场快速检测场景。
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Abstract
Description
Technical Field
[0001] This invention relates to the field of analytical detection technology, and in particular to a self-powered biosensor for detecting aflatoxin B1, its application, and a detection method. Background Technology
[0002] Enzymatic biofuel cells (EBFCs) are devices that directly convert the chemical energy of biomass (such as sugars, alcohols, and lactic acid) into electrical energy. Their core principle is the use of specific redox enzymes as biocatalysts, replacing the precious metal catalysts in traditional fuel cells, to generate electricity under mild conditions (room temperature, atmospheric pressure, and near-neutral pH). This characteristic makes them highly promising for applications in implantable medical devices and portable electronic products.
[0003] Aflatoxin is a highly toxic secondary metabolite produced primarily by fungi, especially *Aspergillus flavus* and *Aspergillus parasiticus*. Due to its serious threat to human health, it has attracted considerable attention. Among its various forms, aflatoxin B1 is the most common, most toxic, and most harmful. Its toxicity can cause irreversible damage to vital organs, particularly the liver, leading to serious health problems. Aflatoxin B1 is widely distributed in nature, especially in food and oil crops. The high temperature and humidity conditions during growth and storage make these crops high-risk contamination areas. Furthermore, the risk of aflatoxin B1 contamination remains during food processing, storage, and transportation, significantly impacting food safety. Therefore, establishing an accurate, rapid, on-site detection technology for mycotoxins is of significant practical importance for protecting public health and promoting sustainable industrial development.
[0004] Currently, rapid detection methods for aflatoxin B1 mainly include immunochromatography, enzyme-linked immunosorbent assay (ELISA), and electrochemical sensing analysis. Colloidal gold immunochromatography and ELISA are the most mature rapid detection methods for aflatoxin B1, with commercially available rapid test kits and strips. However, these commercially available products still exhibit relatively high false-negative and false-positive rates under complex matrix conditions, and most methods are semi-quantitative, failing to achieve rapid and accurate quantification. Furthermore, while electrochemical analysis methods have emerged in recent years, most equipment still relies on external power supplies, complicating on-site rapid detection. Therefore, it is essential to establish a highly selective, sensitive, portable aflatoxin B1 detection method applicable to various environments.
[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention
[0006] The purpose of this invention is to provide a self-powered biosensor for detecting aflatoxin B1, which utilizes a hierarchical porous nanoparticle-modified gold nanoparticle material to fabricate a high-performance enzyme biofuel cell. This overcomes the shortcomings of existing aflatoxin B1 detection methods, such as low accuracy, low sensitivity, inconvenience in portability, and complex operation.
[0007] Another object of the present invention is to provide the use of a self-powered biosensor for detecting aflatoxin B1.
[0008] The present invention also provides a detection method for aflatoxin B1 using a self-powered biosensor.
[0009] To achieve the above objectives, the present invention provides a self-powered biosensor for detecting aflatoxin B1, the self-powered biosensor comprising an anode, a cathode, and an electrolyte; the anode is a bioanode modified with Au@HPC / GOD-cDNA / Apt / CP, and the cathode is a biocathode modified with BOD / Au@HPC / CP; the electrolyte comprises glucose and PBS buffer solution; wherein, the Au@HPC / GOD-cDNA / Apt / CP is obtained by incubating gold nanoparticle-modified hierarchical porous carbon Au@HPC, cDNA, GOD, and Apt; the Au@HPC / CP is obtained by incubating gold nanoparticle-modified hierarchical porous carbon Au@HPC with bilirubin oxidase solution BOD.
[0010] Preferably, in the above technical solution, the preparation of the bioanode includes:
[0011] (1) Au@HPC suspension was drop-coated onto the surface of carbon cloth electrode to prepare Au@HPC / CP;
[0012] (2) The cDNA solution was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide solution and reacted. Then glucose oxidase GOD was added and incubated to obtain GOD-cDNA.
[0013] (3) 6-Mercaptohexyl-1-ol was dropped onto the Au@HPC / CP electrode and incubated. Then, GOD-cDNA was added to the surface of Au@HPC / CP and incubated to obtain Au@HPC / GOD-cDNA / CP.
[0014] (4) Add Aspergillus flavus B1 aptamer Apt to the surface of Au@HPC / GOD-cDNA / CP and incubate to obtain the bioanodide Au@HPC / GOD-cDNA / Apt / CP.
[0015] Preferably, in the above technical solution, step (1) involves adding 30 μL of Au@HPC suspension with a concentration of 1 mg / mL to a 1 cm × 1 cm carbon cloth CP to prepare Au@HPC / CP;
[0016] Preferably, in the above technical solution, in step (2), the amount of cDNA solution added is 100 μL with a concentration of 2 μmol / L; the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide solution added is 100 μL with a concentration of 1 mg / mL, and the mixture is reacted for 1-2 h; the amount of glucose oxidase GOD added is 100 μL with a concentration of 5 mg / mL, and the mixture is incubated at 3-6℃ for 6-8 h;
[0017] Preferably, in the above technical solution, in step (3), the amount of 6-mercaptohexyl-1-ol dripped is 30 μL with a concentration of 1 mmol / L, and incubated for 10-60 min; the amount of GOD-cDNA added is 30 μL, and it is left to stand at 35-40℃ for 10-12 hours.
[0018] Preferably, in the above technical solution, in step (4), the amount of Aspergillus B1 aptamer Apt added is 30 μL with a concentration of 2 μmol / L, and it is incubated at 35-40℃ for 3-4 hours.
[0019] Preferably, in the above technical solution, the preparation of the biocathode includes: drop-coating Au@HPC suspension onto the surface of a carbon cloth electrode to prepare Au@HPC / CP, then dropping bilirubin oxidase solution BOD onto Au@HPC / CP and incubating to obtain the biocathode BOD / Au@HPC / CP.
[0020] Preferably, in the above technical solution, the amount of bilirubin oxidase solution BOD added is 30 μL with a concentration of 5 mg / mL.
[0021] Preferably, in the above technical solution, the preparation of the gold nanoparticle-modified hierarchical porous carbon Au@HPC includes:
[0022] (1) Preparation of hierarchical porous carbon: Sodium alginate was heated to 850-950℃ and stored for 1-2 hours under nitrogen atmosphere. The resulting black solid was soaked in 2-3 mol / L hydrochloric acid solution and stirred. Then it was washed with water, filtered, and dried to obtain hierarchical porous carbon (HPC).
[0023] (2) Preparation of hierarchical porous carbon modified with gold nanoparticles: 0.12 mmol of chloroauric acid was dissolved in 20 mL of water to obtain solution A. 50 mg of polyethylene glycol diacetate was added to 50 mL of HPC aqueous suspension with a concentration of 1 mg / mL. The mixture was sonicated at room temperature for 10-60 min to obtain solution B. Solution A was then slowly added to solution B. The mixture was stirred while maintaining the pH of the mixed solution at 3-5 to obtain Au. 3+ / HPC mixture; then add 10 mL ascorbic acid solution and react for 1-2 h, wherein the molar ratio of ascorbic acid to gold is 3:1; after the reaction is completed, filter, rinse and dry to obtain Au@HPC nanomaterials.
[0024] Preferably, in the above technical solution, the nucleotide sequence of the Aspergillus flavus B1 aptamer Apt is shown in SEQ ID NO:1;
[0025] SEQ ID NO: 1: 5'-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-3'.
[0026] The nucleotide sequence of the cDNA is shown in SEQ ID NO:2;
[0027] SEQ ID NO: 2: 5'-COOH-CCCTTTTGCCTTATATTTTAGACACAGAGAGACAAC AT-3'.
[0028] An application of the above-mentioned self-powered biosensor for detecting aflatoxin B1, said application being for detecting aflatoxin B1.
[0029] A method for detecting aflatoxin B1 using the above-mentioned self-powered biosensor, characterized in that the method includes the following steps:
[0030] (1) Assemble a self-powered biosensor system;
[0031] (2) Measure the instantaneous current value of the self-powered biosensor without introducing Aspergillus flavus B1;
[0032] (3) Plotting the standard curve of Aspergillus flavus B1: First, prepare standard solutions of Aspergillus flavus B1 at different concentrations. Add the standard solutions of Aspergillus flavus B1 at different concentrations to the biocathode BOD / Au@HPC / CP and incubate. Then add exonuclease and incubate. Measure the instantaneous current value of the self-powered biosensor. Plot the standard curve based on the measured current and the corresponding Aspergillus flavus B1 concentration, and then obtain the corresponding linear equation.
[0033] (4) Measure the instantaneous current of the sample to be tested, and substitute it into the linear equation of step (3) to obtain the content of Aspergillus B1 in the sample to be tested.
[0034] Preferably, in the above technical solution, the concentrations of the Aspergillus B1 standard solution are 0, 0.01, 0.1, 1.0, 10, and 10, respectively. 2 10 3 10 4 mg / mL; the linear equation is given in the range of 0.01–10 mg / mL. 4 The linear equation in the pg / mL range is: y = 58.9956lgC + 906.7957 (R 2 =0.9942), where y is the instantaneous current (μA) and C is the concentration of Aspergillus flavus B1 (g / mL); its detection limit is 1.52 fg / mL.
[0035] Compared with existing technologies, the advantages of this invention are as follows: This invention significantly improves electrode conductivity and enzyme loading efficiency through Au@HPC composite materials, and combined with an exonuclease-mediated signal amplification strategy, the detection limit is as low as 1.52 fg / mL, and the linear range covers 0.01–10. 4 The sensitivity is significantly superior to existing detection methods, with a spiked recovery rate of 87.9%–102.8% in actual sample detection, showing high agreement with the results of liquid chromatography, demonstrating excellent accuracy and reliability. In terms of material design, this invention uses hierarchical porous carbon (HPC) as the substrate material. Its interconnected micropores, mesopores, and macropores provide ample loading space for glucose oxidase and bilirubin oxidase, while the macroporous channels facilitate rapid diffusion and mass transfer of substrates and products. In-situ modification with gold nanoparticles not only significantly reduces the charge transfer impedance of the electrode but also enhances the strong immobilization of aptamers and cDNA through Au-S bonds and electrostatic adsorption, thereby greatly improving the stability of the biorecognition interface and signal conversion efficiency. This dual advantage in material structure ensures that the enzyme biofuel cell can output higher and more stable open-circuit voltage and instantaneous current, laying a solid energy foundation for subsequent ultrasensitive detection. Furthermore, the self-powered sensor based on the enzyme biofuel cell requires no external power source, has a simple structure, and is easy to miniaturize, making it particularly suitable for rapid on-site food safety detection scenarios. Attached Figure Description
[0036] Figure 1 This is a schematic diagram of the assembly of the self-powered biosensor for detecting aflatoxin B1 of the present invention;
[0037] Figure 2 This is an electron microscope image of the Au@HPC nanomaterial of this invention;
[0038] in Figure 2a is a scanning electron microscope image of Au@HPC nanomaterials; Figure 2 b is a transmission electron microscope image of Au@HPC nanomaterials;
[0039] Figure 3 The graph shows the instantaneous current response curves under different concentrations of aflatoxin B1.
[0040] Figure 4 It is a graph showing the linear relationship between instantaneous current and the logarithm of aflatoxin B1 concentration;
[0041] Figure 5 These are photographs of instantaneous current values at different aflatoxin B1 concentrations. Detailed Implementation
[0042] The following detailed description of specific embodiments is provided in conjunction with the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.
[0043] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.
[0044] Unless otherwise specified, all raw materials and reagents used in the examples are commercially available. Example
[0045] I. A self-powered biosensor for detecting aflatoxin B1, the self-powered biosensor comprising an anode, a cathode, and an electrolyte; the anode being a bio-anode modified with Au@HPC / GOD-cDNA / Apt / CP, and the cathode being a bio-cathode modified with BOD / Au@HPC / CP; the electrolyte comprising glucose and PBS buffer solution.
[0046] The Au@HPC / GOD-cDNA / Apt / CP is obtained by incubating gold nanoparticle-modified hierarchical porous carbon Au@HPC, cDNA, GOD, and Apt; the Au@HPC / CP is obtained by incubating gold nanoparticle-modified hierarchical porous carbon Au@HPC with bilirubin oxidase solution BOD.
[0047] II. Preparation of a self-powered biosensor for detecting aflatoxin B1
[0048] The preparation of a self-powered biosensor for detecting aflatoxin B1 includes the following steps:
[0049] 1. Preparation of hierarchical porous carbon
[0050] 5g of sodium alginate was placed in a quartz boat and then placed in a tube furnace. The temperature was set to 900℃, the nitrogen flow rate was 3℃ / min, and the material was naturally cooled to obtain carbon material, which was then stored at 900℃ for 1 h. The resulting black solid was immersed in a 3mol / L hydrochloric acid solution and magnetically stirred to remove inorganic impurities, followed by washing with deionized water. The black solid was filtered, collected, and dried at 100℃ for 12 hours. The resulting material is designated as HPC.
[0051] 2. Preparation of hierarchical porous carbon modified with gold nanoparticles
[0052] Dissolve 0.12 mmol of chloroauric acid in 20 mL of deionized water and stir for 30 min to disperse; this solution is named Solution A. Add 50 mg of polyvinylphenylene glycol to 40 mL of (1 mg / mL) HPC aqueous suspension, and sonicate the mixture at room temperature for 30 min; this solution is named Solution B. Slowly add Solution A to Solution B, and then stir at room temperature for 30 min (adjust the pH of the solution to 3-5 using 1 mol / L hydrochloric acid solution) to obtain Au. 3+ A mixture of Au@HPC and ascorbic acid was prepared. Then, 10 mL of ascorbic acid solution (ascorbic acid / gold (molar ratio) = 3:1) was added, and the mixture was reacted at room temperature for 1 h. After the reaction, the product was filtered through a glass frit funnel (2 μm pore size) and washed, first with anhydrous ethanol and then with deionized water, repeated three times, 50 mL each time. Finally, the product was vacuum dried in a constant temperature oven at 60 ℃ for 12 h to obtain Au@HPC nanomaterials. The prepared Au@HPC was prepared into a 1 mg / mL suspension.
[0053] 3. Fabrication of the cathode for the biosensor
[0054] (1) Au@HPC / CP was prepared by dropping 30 μL of Au@HPC suspension (1 mg / mL) onto carbon cloth (CP, 1 cm × 1 cm).
[0055] (2) 30 μL of bilirubin oxidase solution (BOD, 5 mg / mL) was dropped onto Au@HPC / CP and incubated overnight to obtain the biocathode BOD / Au@HPC / CP.
[0056] 4. Fabrication of the anode for the biosensor
[0057] (1) Au@HPC / CP was prepared by dropping 30 μL of Au@HPC suspension (1 mg / mL) onto carbon cloth (CP, 1 cm × 1 cm).
[0058] (2) In an ultrafiltration centrifuge tube, 100 μL of cDNA (2 μmol / L) was mixed with 100 μL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) solution and reacted for 1 h. The EDC / NHS was mixed at a volume ratio of 1:1 and the concentration was 1 mg / mL. Then, the mixture was washed with diethyl pyrocarbonate. After washing, 100 μL (5 mg / mL) of glucose oxidase (GOD) solution was added to the cDNA, and the mixture was mixed for 8 h and incubated at 4 °C. Unbound cDNA was removed by washing with diethyl pyrocarbonate to obtain GOD-cDNA.
[0059] (3) 30 μL of 6-mercaptohexyl-1-ol MCH (1 mmol / L) was dropped onto the Au@HPC / CP electrode and incubated for 30 min. Then, 30 μL of GOD-cDNA was added to the surface of Au@HPC / CP and incubated overnight at 37°C to obtain Au@HPC / GOD-cDNA / CP.
[0060] (4) Inject 30 μL of aflatoxin B1 aptamer Apt (2 μmol / L) onto the Au@HPC / GOD-cDNA / CP electrode and incubate at 37℃ for 140 min. The resulting bioanolyte is named Au@HPC / GOD-cDNA / Apt / CP and stored at 4℃ before use.
[0061] The nucleotide sequence of Aspergillus flavus B1 aptamer Apt is shown in SEQ ID NO:1;
[0062] SEQ ID NO: 1: 5'-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-3'.
[0063] The nucleotide sequence of the cDNA is shown in SEQ ID NO:2;
[0064] SEQ ID NO: 2: 5'-COOH-CCCTTTTGCCTTATATTTTAGACACAGAGAGACAAC AT-3'.
[0065] 5. Prepare electrolytes
[0066] The electrolyte was a 0.1 mol / L phosphate-buffered saline (PBS, pH 7.4) solution containing 5 mmol / L glucose.
[0067] 6. Assembly of a self-powered biosensor system
[0068] according to Figure 1The schematic diagram shows the assembly of a direct-powered biosensor system, using a 100 μF, 25 V capacitor for assembly.
[0069] 7. Preparation of Aflatoxin B1 Standard Solution
[0070] Using water as a solvent, a 1 mg / mL Aspergillus B1 standard solution was diluted to obtain concentrations of 0, 0.01, 0.1, 1.0, 10, and 10 mg / mL. 2 10 3 10 4 mg / mL of Aspergillus B1 standard solution to be tested.
[0071] 8. Construction of the aflatoxin B1 standard curve
[0072] 30 μL of Aspergillus flavus B1 solutions of different concentrations were added dropwise to BOD / Au@HPC / CP and incubated at 37°C for 60 min. Then, 3.6 μL of exonuclease I (ExoⅠ, 2 U / μL) was added, and the mixture was incubated at 37°C for 45 min. Switches a and b were closed, and switch c was opened for 60 s to charge the capacitor. Then, switches b and c were closed, and switch c was opened again. The instantaneous current after charging was measured using a multimeter. A standard curve was plotted based on the measured current versus the logarithm of the corresponding Aspergillus flavus B1 concentration, and the corresponding linear equation was obtained; within the range of 0.01–10... 4 The linear equation in the pg / mL range is: y = 58.9956lgC + 906.7957 (R 2 =0.9942), where y is the instantaneous current (μA) and C is the concentration of Aspergillus flavus B1 (g / mL); its detection limit is 1.52 fg / mL; during detection, the above-mentioned Aspergillus flavus B1 standard solution is replaced with the sample to be tested, and the instantaneous current is substituted into the obtained linear equation to calculate the content of Aspergillus flavus B1 in the sample to be tested. Example
[0073] Perform sample determination
[0074] Take 5 g of rice or peanut sample, weigh it, and transfer it to a 50 mL centrifuge tube. Then add 20 mL of acetonitrile / water (84 / 16, volume ratio) solution. Stir the mixture with 20 oscillations for 20 min, and then centrifuge at 6000 r / min for 10 min. Afterward, collect the supernatant and perform analysis according to the procedure in step 8 of Example 1. Calculate the spiked recovery rate, and the results are shown in Table 1.
[0075] Table 1. Detection and analysis results of Aspergillus flavus B1 in rice and peanuts
[0076]
[0077] As can be seen from Table 1, the determination results of the samples in the method of the present invention are consistent with the determination results of the liquid chromatography method, and the spiked recovery rate of the method of the present invention is above 87.9-102.8%, indicating that the self-powered sensing method proposed in the present invention for detecting Aspergillus flavus B1 has good practicality and accuracy.
[0078] In this invention, Figure 2 a is a scanning electron microscope image of Au@HPC nanomaterials; Figure 2 b is a transmission electron microscope (TEM) image of the Au@HPC nanomaterial. (See image below.) Figure 2 As shown, the HPC surface has a high density of pores. Simultaneously, gold nanoparticles are uniformly distributed on the HPC surface and within the macropores, indicating that the gold nanoparticles have been successfully incorporated into the HPC matrix.
[0079] Figure 3 shows the instantaneous current response under different concentrations of Aspergillus flavus B1; Figure 4 shows the linear relationship between instantaneous current and the logarithm of Aspergillus flavus B1 concentration; Figure 5 shows photographs of instantaneous current values under different Aspergillus flavus B1 concentrations.
[0080] As shown in Figure 3 and Figure 5 As shown in Figure 4, the instantaneous current increases with increasing Aspergillus B1 concentration; Figure 4 shows that in the range of 0.01–10... 4 The instantaneous current value within the pg / mL range showed a good linear relationship with the logarithm of the Aspergillus B1 concentration.
[0081] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.
Claims
1. A self-powered biosensor for detecting aflatoxin B1, characterized in that, The self-powered biosensor includes an anode, a cathode, and an electrolyte; the anode is a bio-anode modified with Au@HPC / GOD-cDNA / Apt / CP, and the cathode is a bio-cathode modified with BOD / Au@HPC / CP; the electrolyte includes glucose and PBS buffer solution. The Au@HPC / GOD-cDNA / Apt / CP is obtained by incubating gold nanoparticle-modified hierarchical porous carbon Au@HPC, cDNA, GOD, and Apt; the Au@HPC / CP is obtained by incubating gold nanoparticle-modified hierarchical porous carbon Au@HPC with bilirubin oxidase solution BOD.
2. The self-powered biosensor for detecting aflatoxin B1 according to claim 1, characterized in that, The preparation of the bioanode includes: (1) Au@HPC suspension was drop-coated onto the surface of carbon cloth electrode to prepare Au@HPC / CP; (2) The cDNA solution was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide solution and reacted. Then glucose oxidase GOD was added and incubated to obtain GOD-cDNA. (3) 6-Mercaptohexyl-1-ol was dropped onto the Au@HPC / CP electrode and incubated. Then, GOD-cDNA was added to the surface of Au@HPC / CP and incubated to obtain Au@HPC / GOD-cDNA / CP. (4) Add Aspergillus flavus B1 aptamer Apt to the surface of Au@HPC / GOD-cDNA / CP and incubate to obtain the bioanodide Au@HPC / GOD-cDNA / Apt / CP.
3. The self-powered biosensor for detecting aflatoxin B1 according to claim 2, characterized in that, Step (1) Add 30 μL of Au@HPC suspension with a concentration of 1 mg / mL to a 1 cm × 1 cm carbon cloth CP to prepare Au@HPC / CP; Step (2): The amount of cDNA solution added is 100 μL with a concentration of 2 μmol / L; the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide solution added is 100 μL with a concentration of 1 mg / mL, and the mixture is reacted for 1-2 h; the amount of glucose oxidase GOD added is 100 μL with a concentration of 5 mg / mL, and the mixture is incubated at 3-6℃ for 6-8 h. Step (3): 30 μL of 6-mercaptohexyl-1-ol was added at a concentration of 1 mmol / L, and incubated for 10-60 min; 30 μL of GOD-cDNA was added and the mixture was left to stand at 35-40℃ for 10-12 hours. Step (4) The amount of Aspergillus B1 aptamer Apt added is 30 μL with a concentration of 2 μmol / L, and it is incubated at 35-40℃ for 3-4 hours.
4. The self-powered biosensor for detecting aflatoxin B1 according to claim 1, characterized in that, The preparation of the biocathode includes: drop-coating Au@HPC suspension onto the surface of a carbon cloth electrode to prepare Au@HPC / CP, then dropping bilirubin oxidase solution BOD onto Au@HPC / CP and incubating to obtain the biocathode BOD / Au@HPC / CP.
5. The self-powered biosensor for detecting aflatoxin B1 according to claim 4, characterized in that, The amount of bilirubin oxidase solution BOD added is 30 μL with a concentration of 5 mg / mL.
6. The self-powered biosensor for detecting aflatoxin B1 according to any one of claims 1-5, characterized in that, The preparation of the hierarchical porous carbon Au@HPC modified with gold nanoparticles includes: (1) Preparation of hierarchical porous carbon: Sodium alginate was heated to 850-950℃ and stored for 1-2 hours under nitrogen atmosphere. The resulting black solid was soaked in 2-3 mol / L hydrochloric acid solution and stirred. Then it was washed with water, filtered, and dried to obtain hierarchical porous carbon (HPC). (2) Preparation of hierarchical porous carbon modified with gold nanoparticles: 0.12 mmol of chloroauric acid was dissolved in 20 mL of water to obtain solution A. 50 mg of polyethylene glycol diacetate was added to 50 mL of HPC aqueous suspension with a concentration of 1 mg / mL. The mixture was sonicated at room temperature for 20-30 min to obtain solution B. Solution A was then slowly added to solution B. The mixture was stirred while maintaining the pH of the mixed solution at 3-5 to obtain Au. 3+ / HPC mixture; then add 10 mL ascorbic acid solution and react for 1-2 h, wherein the molar ratio of ascorbic acid to gold is 3:1; after the reaction is completed, filter, rinse and dry to obtain Au@HPC nanomaterials.
7. The self-powered biosensor for detecting aflatoxin B1 according to claim 1, characterized in that, The nucleotide sequence of Aspergillus flavus B1 aptamer Apt is shown in SEQ ID NO:1; The nucleotide sequence of the cDNA is shown in SEQ ID NO:
2.
8. The application of a self-powered biosensor for detecting aflatoxin B1 as described in claim 1, characterized in that, The application is for detecting aflatoxin B1.
9. A method for detecting aflatoxin B1 using a self-powered biosensor as described in claim 1, characterized in that, The method includes the following steps: (1) Assemble a self-powered biosensor system; (2) Measure the instantaneous current value of the self-powered biosensor without introducing Aspergillus flavus B1; (3) Plotting the standard curve of Aspergillus flavus B1: First, prepare standard solutions of Aspergillus flavus B1 at different concentrations. Add the standard solutions of Aspergillus flavus B1 at different concentrations to the biocathode BOD / Au@HPC / CP and incubate. Then add exonuclease and incubate. Measure the instantaneous current value of the self-powered biosensor. Plot the standard curve based on the measured current and the corresponding Aspergillus flavus B1 concentration, and then obtain the corresponding linear equation. (4) Measure the instantaneous current of the sample to be tested, and substitute it into the linear equation of step (3) to obtain the content of Aspergillus B1 in the sample to be tested.
10. The method according to claim 9, characterized in that, The concentrations of the Aspergillus B1 standard solution were 0, 0.01, 0.1, 1.0, 10, and 10⁻⁶. 2 10 3 10 4 mg / mL; The linear equation is given by equation (0.01–10). 4 The linear equation in the pg / mL range is: y = 58.9956lgC + 906.7957 (R 2 =0.9942), where y is the instantaneous current (μA) and C is the concentration of Aspergillus flavus B1 (g / mL); its detection limit is 1.52 fg / mL.