Polya-mediated label-free electrochemical sensor, preparation method and application thereof

Abstract

The application provides a PolyA-mediated label-free electrochemical sensor and a preparation method and application thereof, and relates to the technical field of electrochemical biosensing. The sensor adopts a three-electrode system as a whole working system, the three-electrode system comprises a working electrode, a reference electrode and an auxiliary electrode; the working electrode is a PolyA-mediated label-free electrochemical aptamer sensor, an AFB1 aptamer is used as a biological recognition element, the biological recognition element is paired with a DNA segment containing a PolyA sequence in a complementary manner, and then is fixed on the surface of a gold electrode by the specific affinity between PolyA and gold; the reference electrode is a silver chloride electrode, and the auxiliary electrode is a platinum wire electrode. The PolyA-mediated label-free electrochemical sensor has the advantages of high detection sensitivity, strong specificity, simple operation, fast detection speed and low cost.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensing technology, specifically to a PolyA-mediated label-free electrochemical sensor, its preparation method, and its application. Background Technology

[0002] Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic secondary metabolite produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. It has a broad spectrum of contamination and is prevalent in grain and oil products, nuts, feed, and animal-derived foods. Food is easily contaminated by fungi in the environment during production, processing, and storage, leading to AFB1 residues. AFB1 contamination not only reduces the quality of food and feed but also accumulates through the food chain, posing a serious threat to livestock farming efficiency, human health, and public health safety.

[0003] AFB1 is one of the most potent known mycotoxins, 10 times more toxic than cyanide and 68 times more toxic than arsenic. It is also highly carcinogenic, primarily damaging the liver tissue of humans and animals. Long-term consumption of peanuts and other foods containing AFB1 residues can lead to chronic hepatitis, cirrhosis, and even liver cancer. It may also damage the immune and reproductive systems, with infants and the elderly being particularly vulnerable. Furthermore, AFB1 residues can affect food import and export trade and restrict the healthy development of related industries. Therefore, establishing a rapid, sensitive, and accurate method for detecting AFB1 residues in peanuts is of significant practical importance and application value for standardizing food production and processing procedures, ensuring food safety, protecting human health, and promoting the high-quality development of related industries.

[0004] Currently, existing technologies for detecting AFB1 residues mainly include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). While instrumental analytical methods such as high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) offer advantages such as high detection precision, good accuracy, strong anti-interference capabilities, and low detection limits, meeting the needs of precise laboratory testing and trace analysis, they also have significant technical drawbacks: First, the sample pretreatment process is cumbersome and complex. Food sample matrices are complex, requiring multiple steps such as extraction, purification, concentration, and derivatization before testing, which is difficult to operate, time-consuming, and prone to sample loss, affecting the accuracy of the test results. Second, the testing cycle is long, with a single test taking several hours, making it impossible to achieve rapid screening of large batches of samples or on-site emergency testing. Third, the testing instruments and equipment are expensive, bulky, and have high maintenance costs. They also have stringent requirements for the operating environment and the professional skills of operators, requiring professional personnel to undergo systematic training before they can operate them proficiently. This makes them unsuitable for the rapid testing needs of food production enterprises, farmers' markets, and inspection and quarantine sites, limiting their promotion and application in routine testing and on-site supervision of food and agricultural product quality and safety.

[0005] While enzyme-linked immunosorbent assay (ELISA) has advantages such as relatively simple operation, moderate detection cost, and the ability to perform batch detection, and has been used to some extent in grassroots testing, this method has shortcomings such as insufficient sensitivity and poor specificity. It is easily affected by other components in the food matrix, resulting in false positive and false negative results. It cannot meet the requirements for accurate detection of trace AFB1 residues. In addition, the detection process requires the use of specific antibodies, which have a long preparation cycle, poor stability, and are easily affected by environmental factors such as temperature and humidity, further limiting its detection performance and application range.

[0006] In recent years, electrochemical sensing technology has become a research hotspot in food safety testing, environmental monitoring, and biomedical detection due to its outstanding advantages such as simple operation, fast detection speed, miniaturized instruments, low cost, high sensitivity, and no need for complex pretreatment. It has been widely used in the rapid detection of various mycotoxins, pesticide residues, and veterinary drug residues. Among them, electrochemical aptamer sensors have shown good application potential in AFB1 residue detection due to their advantages such as high specificity, good stability, easy synthesis and modification of aptamers, and reusability, and are expected to solve the technical defects of existing detection methods. However, the immobilization strategies of existing conventional electrochemical aptamer sensors mostly adopt covalent bonding (such as Au-S bond bonding, amide bond bonding, etc.). This type of immobilization requires the use of precious metal materials (such as gold electrodes, platinum electrodes) and special chemical reagents, resulting in high sensor preparation costs and relatively complex preparation processes. This makes it difficult to meet the application requirements of large-scale, low-cost, and field-based food and agricultural product quality and safety testing, thus limiting its industrialization and grassroots application.

[0007] In summary, existing methods for detecting AFB1 residues either suffer from high detection costs, complex operations, cumbersome pretreatment, long detection cycles, and an inability to achieve rapid on-site detection, or while possessing advantages in rapid or batch detection, they suffer from insufficient sensitivity, poor specificity, high preparation costs, and poor stability. Neither approach adequately meets the needs for rapid, accurate, and low-cost on-site detection of trace AFB1 residues in food, and fails to effectively solve the challenges of AFB1 residue detection in food production, distribution, and regulatory processes. Therefore, developing a low-cost, highly sensitive, highly specific, easy-to-operate method and related apparatus for rapid on-site detection of AFB1 residues in food, without complex pretreatment, to overcome the aforementioned technical deficiencies of existing technologies, has become a pressing technical challenge in this field and is the starting point and core objective of this invention. Summary of the Invention

[0008] This invention relates to the field of electrochemical biosensing technology, specifically to a PolyA-mediated label-free electrochemical sensor, its preparation method, and its application. The aim is to address the shortcomings of existing AFB1 detection methods, such as low sensitivity, poor specificity, complex operation, low detection efficiency, or high cost, and to achieve rapid and accurate quantitative detection of AFB1 in peanut samples.

[0009] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0010] The first aspect of this invention provides a PolyA-mediated label-free electrochemical sensor employing a three-electrode system as the overall working system. The three-electrode system includes a working electrode, a reference electrode, and an auxiliary electrode. The working electrode is a PolyA-mediated label-free electrochemical aptamer sensor, using the AFB1 aptamer as a biorecognition element. This biorecognition element pairs complementaryly with a DNA fragment containing the PolyA sequence and is then immobilized on the gold electrode surface by the specific affinity of PolyA for gold, requiring no additional labeling. The reference electrode is a silver chloride electrode, and the auxiliary electrode is a platinum wire electrode. The sequence of the AFB1 aptamer includes: 5′-GGG TGG GTG GGT GGG GTT GGG CAC GTG TTG TCT CTC TGTGTC TCG TGC CCT TCG CTA GGC CCA CA-3′ and 5′-ACA CCC GGA TCG CTT CCC GTG CTCTGT GTC TCT CTG TTG TGC ACG GGT TG GGG TGG GTG GGT GGG-3', the sequence of PolyA is: 5′-TGT GGG CCT AGC GAA GGG CAC GAG ACA CAG AGA GAC AAC ACG TGC CCA AC TTT TTTA10 TTT TTT CAA CCC GTG CAC AAC AGA GAG ACA CAG AGC ACG GGA AGC GAT CCG GGTGT-3′.

[0011] The second aspect of this invention provides a method for preparing the above-mentioned PolyA-mediated label-free electrochemical sensor, comprising the following steps: (1) Pretreatment of working electrode: Using Au electrode as base electrode, the electrode is polished with alumina slurry, ultrasonically cleaned with anhydrous ethanol and first-grade water, electrochemically cleaned with 0.6 M H2SO4 solution, rinsed with deionized water and dried with nitrogen gas flow, and then set aside for use. (2) Folding of AFB1 aptamer to form G-quadruplex: AFB1 aptamer was prepared into a 150 μM stock solution, and 10 mM K was added. + The folding buffer was diluted to a final concentration of 6 μM, and then subjected to heat denaturation at 95 °C for 5 min and annealing at 25 °C for 4 h for later use. (3) Sensor construction: S1. Drop 1.0 μM, 10 μL PolyA solution onto the pretreated Au electrode surface, incubate overnight at 25°C, rinse with TE buffer, and dry with nitrogen gas flow; S2. The 10 μM, 10 μLAFB1 aptamer G-tetrachain solution prepared in step (2) was incubated at 25°C for 60 min, rinsed with TE buffer, and dried with nitrogen gas. S3. Drop 10 μL of 0.5 mM mercaptohexanol (MCH) solution onto the substrate, incubate at 25 °C for 45 min, rinse with TE buffer, and dry with nitrogen gas. S4. Incubate in 10 μL of 800 μM methylene blue (MB) solution for 80 min, rinse with TE buffer, and dry with nitrogen gas to obtain the product; The buffer formulation is as follows: TE buffer contains 50 mM Tris-HCl, 1.0 mM EDTA, pH=7.4; K + The folding buffer contains 50 mM Tris-HCl, 100 mM KCl, and 0.1 mM EDTA, with a pH of 7.4.

[0012] Further, in step (1), polishing is performed sequentially using 0.2 μm and 0.05 μm alumina slurries, ultrasonic cleaning is performed for 5 min each time, and electrochemical cleaning is performed until a stable cyclic voltammogram is obtained.

[0013] Further, in step (1), the polishing of the alumina slurry specifically involves: placing the gold electrode on a polishing cloth, adding a small amount of alumina slurry, and gently rotating the electrode by hand to polish the electrode surface evenly until the electrode surface exhibits a uniform mirror gloss, free of scratches and stains; after each polishing, immediately rinse the electrode surface with deionized water; the ultrasonic cleaning specifically involves: placing the polished gold electrode into anhydrous ethanol and deionized water in sequence for ultrasonic cleaning, with the ultrasonic power controlled at 200 W; after ultrasonic cleaning, remove the electrode and rinse it repeatedly with deionized water 3-5 times to ensure that there are no residual reagents on the electrode surface; the electrochemical cleaning specifically involves: using the ultrasonically cleaned gold electrode as the working electrode, the silver chloride electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode to form a three-electrode system, and placing it in a 0.6 M H2SO4 solution; using cyclic voltammetry (CV) to perform electrochemical cleaning of the gold electrode, with the scanning range set to -0.2 V to 1.5 V and the scanning rate at 50 Hz. mV / s ensures that the electrode surface is clean, free of impurities, and has uniform electrochemical performance. Then, rinse the electrochemically cleaned gold electrode with plenty of deionized water 3-5 times, and slowly blow nitrogen gas onto the electrode surface until the electrode is completely dry. Place it in a desiccator for later use.

[0014] Furthermore, the specific operation in step (3) is as follows: S1. PolyA Immobilization: Take the pretreated and dried gold electrode, accurately pipette 10 μL of 1.0 μM PolyA solution, and uniformly drop it onto the working surface of the gold electrode; incubate the electrode overnight at 25 ℃. Utilizing the specific affinity of PolyA molecules for the gold electrode surface, PolyA is firmly immobilized on the gold electrode surface, forming a stable PolyA-modified layer; after incubation, gently rinse the electrode surface three times with TE buffer, each rinse lasting 10 seconds, using gentle pressure to avoid damaging the PolyA-modified layer, thus removing loosely bound and not firmly immobilized PolyA from the electrode surface; then slowly purge the electrode surface with nitrogen gas until completely dry, obtaining the PolyA-modified gold electrode.

[0015] S2. Aptamer binding: First, prepare a 150 μM stock solution of AFB1 aptamer, then add 10 mM K. + Folding buffer was used to dilute and bring the aptamer concentration to a final level of 6 μM. The aptamer was then subjected to heat denaturation at 95 °C for 5 min and annealing at 25 °C for 4 h to fold into a G-quadruplex with specific AFB1 recognition capability. Using a pipette, 10 μL of the folded AFB1 aptamer solution was evenly applied to the electrode surface of the modified electrode (S1) and incubated at 25 °C for 60 min. This allowed the aptamer to firmly bind to the PolyA-modified electrode surface through complementary pairing with the PolyA sequence. After incubation, the electrode surface was rinsed three times with TE buffer to remove uncomplementary free aptamers. The electrode was then purged with nitrogen and dried to obtain the aptamer-modified electrode.

[0016] S3. Non-specific site blocking: Take the aptamer-modified electrode obtained in S2, and use a pipette to draw 10 μL of 0.5 mM MCH solution, and evenly drop it onto the electrode surface. Incubate at 25 ℃ for 45 min. The thiol groups in the MCH molecule bind to the residual active sites of unbound PolyA on the gold electrode surface, blocking the non-specific binding sites on the electrode surface. This effectively avoids the non-specific adsorption of aptamers, AFB1, and signal molecules, reducing background interference. After incubation, rinse the electrode surface three times with TE buffer to remove unbound free MCH, and dry it with nitrogen to obtain the blocked modified electrode.

[0017] S4. MB Incubation: Take the modified electrode blocked by MCH in S3, pipette 10 μL of 800 μM MB solution, and evenly drop it onto the electrode surface. Incubate at 25 ℃ for 80 min to allow MB molecules to specifically bind to the aptamer G-quadruplex, serving as an electrochemical response signal molecule. After incubation, rinse the electrode surface three times with TE buffer to remove unbound free MB, and slowly purge and dry with nitrogen to finally prepare the PolyA-mediated label-free electrochemical sensor. Store the prepared sensor in a sealed container at 4℃ for up to 10 days.

[0018] A third aspect of the present invention provides an application of the label-free electrochemical aptamer sensor based on the above-described PolyA-mediated strategy for AFB1 detection.

[0019] Furthermore, the steps of the AFB1 detection application include incubating the sensor with the peanut sample solution, and then using square wave stripping voltammetry (SWV) to detect the electrochemical response signal, with the peak current difference ΔI reflecting the AFB1 concentration, thereby achieving quantitative detection of AFB1.

[0020] Furthermore, the detected AFB1 concentration range is 0.001~1000 nM, the linear correlation coefficient R²≥0.995, and the limit of detection is 0.22 pM.

[0021] Furthermore, peanut samples were treated using a five-fold matrix dilution method, and incubated at a constant temperature of 37 ℃ for 60 min. The SWV detection parameters were a scanning range of -0.5 V to 0.5 V and a scanning rate of 25 mV / s.

[0022] The beneficial effects of this invention are as follows: The PolyA-mediated label-free electrochemical sensor provided by this invention has the advantages of high detection sensitivity, strong specificity, simple operation, fast detection speed, and low cost. The sensitivity enhancement effect has been experimentally verified, specifically: within the AFB1 concentration range of 0.001~1000 nM, the sensor's electrochemical response signal (peak current) exhibits a good linear relationship with the AFB1 concentration (correlation coefficient R). 2With a sensitivity ≥0.995, excellent linear response, and strong ability to detect minute changes in AFB1 concentration, its detection sensitivity is more than 50% higher than that of conventional electrochemical aptamer sensors and significantly higher than that of traditional detection methods such as high performance liquid chromatography. It can be directly applied to the rapid and accurate quantitative detection of AFB1 in food samples without the need for complex sample pretreatment steps, adapting to the needs of actual detection scenarios. Even under the interference of complex food matrices, it can still maintain high detection sensitivity with no significant increase in the detection limit. It effectively solves the problem of sensitivity decline in actual sample detection of existing technologies, providing an efficient and practical technical means for AFB1 detection. Attached Figure Description

[0023] Figure 1 The SWV spectra of the PolyA-mediated label-free electrochemical sensor prepared in this invention at different AFB1 standard solution concentrations are shown.

[0024] Figure 2 middle Figure 2 (A) is a graph showing the relationship between AFB1 concentration and ΔI value; Figure 2 (B) AFB1 determination standard curve. Detailed Implementation

[0025] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0026] A method for preparing a PolyA-mediated label-free electrochemical sensor includes the following steps: (1) Pretreatment of working electrode The Au electrode was used as the substrate electrode for cleaning, and the specific procedures were as follows: The surface of the Au electrode was polished sequentially with 0.2 μm and 0.05 μm alumina slurries until the electrode surface was smooth and free of scratches; then the polished Au electrode was placed in anhydrous ethanol and first-stage water, respectively, and ultrasonically cleaned for 5 min each to remove polishing agent residue and impurities adhering to the electrode surface; after cleaning, the Au electrode was placed in 0.6 M H2SO4 solution for electrochemical cleaning until a stable cyclic voltammogram was obtained to ensure uniform electrochemical performance of the electrode surface; finally, the electrode surface was rinsed with a large amount of first-stage deionized water and dried with a nitrogen stream for later use.

[0027] (2) The AFB1 aptamer folds to form a G-quadruplex. Prepare a 150 μM stock solution using AFB1 aptamer, and add 10 mM K to the aptamer solution. +Folding buffer was used to dilute and bring the volume to a final aptamer concentration of 6 μM. The above mixture was placed in a 95 °C metal bath and heated for 5 min for heat denaturation treatment to completely unwind the aptamer single strands. After heat denaturation, the solution was quickly transferred to a 25 °C room temperature environment and allowed to stand for 4 h for renaturation treatment to allow the aptamer to fold efficiently into a G-quadruplex with specific AFB1 recognition ability, which was then ready for use.

[0028] (3) Construction of a label-free electrochemical aptamer sensor mediated by PolyA S1. Take the Au electrode that has been pretreated in step (1), drop a 1.0 μM, 10 μL PolyA solution onto the clean Au electrode surface, and incubate overnight at 25 ℃ to allow PolyA to be stably fixed on the Au electrode surface through its affinity for gold. After incubation, rinse the electrode surface 3-5 times with TE buffer to remove loosely bound PolyA, and then dry the electrode with a stream of nitrogen for later use. S2. Take the Au electrode after the treatment in step S1, and drop the 10 μM, 10 μL AFB1 aptamer G-quadruplex mixed solution prepared in step (2) onto the electrode surface. Incubate at room temperature of 25 ℃ for 60 min to allow the aptamer to bind stably to the electrode surface through complementary pairing with PolyA. After incubation, rinse the electrode surface with TE buffer 3-5 times to remove unbound aptamers, and dry with nitrogen gas for later use. S3. Take the Au electrode treated in step S2, drop 10 μL of 0.5 mM mercaptohexanol (MCH) solution onto the electrode surface, and incubate at 25 ℃ for 45 min. The MCH blocks the active sites of unbound PolyA on the electrode surface, reducing the non-specific binding of subsequent aptamers. After incubation, rinse the electrode surface 3-5 times with TE buffer to remove unbound MCH, and dry with nitrogen gas for later use. S4. Take the Au electrode treated in step S3 and place it in a 10 μL solution of methylene blue (MB) with a concentration of 800 μM. Incubate for 80 min to allow MB to bind to the aptamer G-quadruplex as an electrochemical signal molecule. After incubation, rinse the electrode surface with TE buffer 3-5 times to remove unbound MB and dry it with nitrogen gas to obtain the PolyA-mediated label-free electrochemical aptamer sensor.

[0029] The above TE buffer formulation is as follows: containing 50 mM Tris-HCl, 1.0 mM EDTA, and pH adjusted to 7.4; K +The folding buffer formulation is as follows: containing 50 mM Tris-HCl, 100 mM KCl, and 0.1 mM EDTA, with the pH adjusted to 7.4.

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0031] Description of reagents and materials used in this embodiment: 1. Aptamers and PolyA sequences: The aptamers for AFB1 are: 5′-GGG TGG GTG GGT GGG GTTGGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-3′ and 5′-ACACCC GGA TCG CTT CCC GTG CTC TGT GTC TCT CTG TTG TGC ACG GGT TG GGG TGG GTGGGT GGG-3′; The sequence for PolyA is: 5′-TGT GGG CCT AGC GAA GGG CAC GAG ACA CAG AGA GACAAC ACG TGC CCA AC TTT TTT A10 TTT TTT CAA CCC GTG CAC AAC AGA GAG ACA CAGAGC ACG GGA AGC GAT CCG GGT GT-3′. The aptamers and PolyA sequences mentioned above were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) with a purity of ≥98%.

[0032] 2. Buffer formulation: The TE buffer contains 50 mM Tris-HCl, 1.0 mM EDTA, pH=7.4; the K... + The folding buffer contains 50 mM Tris-HCl, 100 mM KCl, and 0.1 mM EDTA, with a pH of 7.4; the PBS buffer electrolyte solution has a pH of 7.4. Both are prepared with analytical grade reagents and filtered through a 0.22 μm filter membrane to remove impurities before use.

[0033] 3. Other reagents: alumina slurry (0.2 μm, 0.05 μm), anhydrous ethanol, deionized water (grade I), MCH (mercaptohexanol), MB (methylene blue), and AFB1 standard (purity ≥99%), all of which were analytical grade; commercially available peanuts uncontaminated with AFB1 were purchased from a local supermarket.

[0034] 4. Instruments and equipment: electrochemical workstation, three-electrode system (gold electrode as working electrode substrate, silver chloride electrode as reference electrode, platinum wire electrode as auxiliary electrode), pipette, metal bath, ultrasonic cleaner, nitrogen cylinder, electronic balance, 4℃ refrigerator, etc.

[0035] Example 1: Fabrication of a PolyA-mediated label-free electrochemical aptamer sensor (1) Pretreatment of working electrode S1. Polishing treatment: Place the gold electrode on a polishing cloth, and drop small amounts of 0.2 μm and 0.05 μm alumina paste onto it respectively. Gently rotate the electrode by hand to polish the electrode surface evenly until the electrode surface presents a uniform mirror gloss, without scratches or stains. After each polishing, immediately rinse the electrode surface with deionized water to thoroughly remove residual alumina paste and avoid residual impurities affecting the subsequent modification effect.

[0036] S2. Ultrasonic cleaning: Place the polished gold electrode into anhydrous ethanol and deionized water in sequence, and ultrasonically clean for 5 minutes each. The ultrasonic power is controlled at 200 W to fully remove organic matter, polishing agent residue and other impurities adsorbed on the electrode surface. After ultrasonic cleaning, remove the electrode and rinse it repeatedly with deionized water 3 to 5 times to ensure that there is no residual reagent on the electrode surface.

[0037] S3. Electrochemical Cleaning: The ultrasonically cleaned gold electrode was used as the working electrode, the silver chloride electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode, forming a three-electrode system, which was placed in a 0.6 M H2SO4 solution. Cyclic voltammetry (CV) was used to electrochemically clean the gold electrode, with the scan range set to -0.2 V to 1.5 V and the scan rate set to 50 mV / s, to ensure that the electrode surface was clean, free of impurities, and with uniform electrochemical performance. Subsequently, the electrochemically cleaned gold electrode was rinsed with a large amount of deionized water 3 to 5 times, and the electrode surface was slowly purged with nitrogen until the electrode was completely dry, and then placed in a desiccator for later use.

[0038] (2) The AFB1 aptamer folds to form a G-quadruplex. Prepare a 150 μM stock solution using AFB1 aptamer, and add 10 mM K to the aptamer solution. + Folding buffer was used to dilute and bring the volume to a final aptamer concentration of 6 μM. The above mixture was placed in a 95 °C metal bath and heated for 5 min for heat denaturation treatment to completely unwind the aptamer single strands. After heat denaturation, the solution was quickly transferred to a 25 °C room temperature environment and allowed to stand for 4 h for renaturation treatment to allow the aptamer to fold efficiently into a G-quadruplex with specific AFB1 recognition ability, which was then ready for use.

[0039] (3) Construction of label-free electrochemical aptamer sensors S1. PolyA Immobilization: Take the pretreated and dried gold electrode described above, and accurately pipette 10 μL of 1.0 μM PolyA solution, uniformly drop it onto the working surface of the gold electrode, avoiding overflow of the solution. Incubate the electrode overnight (12-16 h) at a constant temperature of 25 ℃. Utilizing the specific affinity of PolyA molecules for the gold electrode surface, PolyA is firmly immobilized on the gold electrode surface, forming a stable PolyA-modified layer. After incubation, gently rinse the electrode surface three times with TE buffer, each rinse lasting 10 s, using gentle pressure to avoid damaging the PolyA-modified layer, in order to remove loosely bound and not firmly immobilized PolyA from the electrode surface. Then, slowly purge the electrode surface with nitrogen gas until completely dry to obtain the PolyA-modified gold electrode.

[0040] S2. Aptamer binding: First, prepare a 150 μM stock solution of AFB1 aptamer, then add 10 mM K. + Folding buffer was used to dilute and bring the aptamer concentration to a final level of 6 μM. The aptamer was then subjected to heat denaturation at 95 °C for 5 min and annealing at 25 °C for 4 h to fold into a G-quadruplex with specific AFB1 recognition capability. Using a pipette, 10 μL of the folded AFB1 aptamer solution was evenly applied to the electrode surface of the modified electrode (S1) and incubated at 25 °C for 60 min. This allowed the aptamer to firmly bind to the PolyA-modified electrode surface through complementary pairing with the PolyA sequence. After incubation, the electrode surface was rinsed three times with TE buffer to remove uncomplementary free aptamers. The electrode was then purged with nitrogen and dried to obtain the aptamer-modified electrode.

[0041] S3. Non-specific site blocking: Take the aptamer-modified electrode obtained in S2, and use a pipette to draw 10 μL of 0.5 mM MCH solution, and evenly drop it onto the electrode surface. Incubate at 25 ℃ for 45 min. The thiol groups in the MCH molecule bind to the residual active sites of unbound PolyA on the gold electrode surface, blocking the non-specific binding sites on the electrode surface. This effectively avoids the non-specific adsorption of aptamers, AFB1, and signal molecules, reducing background interference. After incubation, rinse the electrode surface three times with TE buffer to remove unbound free MCH, and dry it with nitrogen to obtain the blocked modified electrode.

[0042] S4. MB Incubation: Take the modified electrode blocked by MCH in S3, pipette 10 μL of 800 μM MB solution, and evenly drop it onto the electrode surface. Incubate at 25 ℃ for 80 min to allow MB molecules to specifically bind to the aptamer G-quadruplex, serving as an electrochemical response signal molecule. After incubation, rinse the electrode surface three times with TE buffer to remove unbound free MB, and slowly purge and dry with nitrogen to finally prepare the PolyA-mediated label-free electrochemical sensor. Store the prepared sensor in a sealed container at 4℃ for up to 10 days.

[0043] Detection example: Detection of AFB1 in peanuts using a PolyA-mediated label-free electrochemical sensor prepared in Example 1. This detection example uses the PolyA-mediated label-free electrochemical sensor prepared in Example 1. Its detection capability for AFB1 in peanuts is investigated using electrochemical detection methods, focusing on verifying the sensor's sensitivity, specificity, and stability. Different concentrations of AFB1 specifically bind to the aptamers on the sensor surface, causing regular changes in the sensor's electrochemical response signal (peak current). This example uses square wave stripping voltammetry (SWV) for detection, recording the redox peak current values. The peak current difference reflects the AFB1 concentration. Specific steps are as follows: (1) Sensor sensitivity detection S1. Blank signal detection: The PolyA-mediated label-free electrochemical aptamer sensor prepared in Example 1 was placed in a PBS buffer electrolyte solution with a pH of 7.4 and detected using SWV. The detection parameters were set as follows: scan range of -0.5 V to 0.5 V, scan rate of 25 mV / s, and the peak current values ​​of the redox peak of the sensor were recorded. I a (blank signal value).

[0044] S2. Standard Solution Incubation and Detection: AFB1 concentrations of 0.001, 0.005, 0.01, 0.10, 1.0, 10.0, 100.0, 300.0, 500.0, and 1000 nM were prepared. The prepared electrochemical aptamer sensor was placed in the above AFB1 standard solutions of different concentrations and incubated at 37 ℃ for 60 min to allow AFB1 to fully and specifically bind to the aptamer on the sensor surface. After incubation, the sensor surface was gently rinsed three times with TE buffer solution at pH 7.4 to remove unbound free AFB1. The sensor surface was then slowly dried with nitrogen gas to obtain the PolyA-mediated label-free electrochemical aptamer sensor after incubation with AFB1.

[0045] S3. Response signal detection and standard curve establishment: Sensors incubated with different concentrations of AFB1 were placed in PBS buffer electrolyte solution with a pH of 7.4 and detected using the same SWV parameters as described above. The peak current Ib of the redox peak of each sensor was obtained. The peak current difference ΔI = Ia - Ib before and after AFB1 incubation was calculated. The higher the AFB1 concentration, the more it binds to the aptamer, resulting in fewer MB molecules bound to the electrode surface, a smaller peak current Ib, and a larger peak current difference ΔI.

[0046] Figure 1 The SWV spectra of the PolyA-mediated label-free electrochemical sensor at different AFB1 standard solution concentrations are shown in the figure. It can be clearly seen from the figure that as the AFB1 concentration increases, the redox peak current of the sensor gradually decreases, while the peak shape remains stable. Figure 2 middle, Figure 2 A is a graph showing the relationship between AFB1 concentration and ΔI value, indicating the correlation between AFB1 concentration and peak current difference ΔI. I There is a clear positive correlation; Figure 2 B is the standard curve for AFB1 determination, using the logarithm of AFB1 concentration (lg(c)). AFB1 Using / nM)) as the x-axis and the peak current difference ΔI as the y-axis, an AFB1 detection standard curve is established, with the regression equation being: ΔI=13.55+3.03×lg(c AFB1 / nM), correlation coefficient R² =0.995.

[0047] Experimental results show that the PolyA-mediated label-free electrochemical sensor exhibits good linearity and excellent linear response in the AFB1 concentration range of 0.001–1000 nM. According to the 3σ rule, the limit of detection (LOD) of the sensor for AFB1 is 0.22 pM, demonstrating extremely high detection sensitivity. It can effectively capture trace AFB1 residues and solve the technical pain point of insufficient sensitivity in existing technologies.

[0048] (2) Sensor-specific detection Other common antibiotics found in peanuts were selected as interfering substances, and standard solutions of each interfering substance with the same concentration (150 nM) as AFB1 were prepared. Following the above sensitivity detection steps, the PolyA-mediated label-free electrochemical sensor prepared in Example 1 was incubated with each interfering substance standard solution and the AFB1 standard solution, respectively. The response signal to the interfering substance was only 3.2% of that to the target substance, indicating that the AFB1 aptamer of the PolyA-mediated label-free electrochemical aptamer sensor exhibits good specificity only with AFB1.

[0049] (3) Sensor stability detection Multiple sets of sensors prepared in Example 1 were sealed and stored in a refrigerator at 4 ℃. After storage for 0, 2, 4, 6, 8 and 10 days, the measured sensor current difference values ​​showed a SWV signal retention rate of 93.5% and an RSD of 2.6% compared to before storage (0 days). This indicates that the sensor constructed in this invention has good stability and can meet the requirements for long-term storage and use in actual detection.

[0050] Verification example: The recovery rate test using the standard addition method verifies the practical application capability of this sensor. This validation example uses a standard addition method recovery test to verify the accuracy and practicality of the PolyA-mediated label-free electrochemical sensor of this invention in detecting AFB1 in actual peanut samples. A comparative validation is also performed using national standard detection methods. The specific steps are as follows: (1) Sample preparation Commercially available peanuts uncontaminated with AFB1 were taken and pretreated to obtain the sample solution to be tested. A certain volume of AFB1 standard solution was added to each sample to make the AFB1 spiking concentration in the peanuts reach 29.2 nM and 58.4 nM, respectively. The mixture was stirred thoroughly and allowed to stand for 30 min to allow AFB1 to be evenly distributed in the peanuts. Each peanut sample with a spiking concentration was divided into two parts. One part was used for detection by the sensor constructed in this invention, and the other part was used for detection according to the national standard method. The measurements were performed in parallel for 3 times, and the average value was taken.

[0051] (2) Sample testing S1. Sensor Detection of the Present Invention: The peanut samples containing AFB1 were treated using a five-fold matrix dilution method to reduce the interference of the peanut matrix on detection. The aptamer sensor prepared in Example 1 was incubated with the treated peanut samples at a constant temperature of 37 ℃ for 60 min. After incubation, the sensor surface was rinsed three times with TE buffer to remove residual sample matrix and unbound AFB1. Detection was performed using the SWV method, and the peak current values ​​Ia and Ib were recorded. ΔI was calculated, and the amount of AFB1 detected in the peanut samples was calculated using the standard curve established in Example 2. The recovery rate and relative standard deviation (RSD) were 98.2%–103.4% and 2.5–3.4%, respectively. The HPLC-MS / MS detection recovery rate was 85.3%–114.2%, and the RSD value was 3.7–4.9%. This demonstrates that the PolyA-mediated label-free electrochemical aptamer sensor prepared in this invention has superior accuracy compared to the HPLC-MS / MS method, and requires no expensive instruments or complex pretreatment. It is simple to operate, low in cost, and more suitable for large-scale, rapid on-site screening of AFB1 residues in peanuts.

[0052] Summary of Implementation Examples In summary, the PolyA-mediated label-free electrochemical biosensor of this invention employs a three-electrode system, using a gold electrode as the working electrode substrate and an aptamer of AFB1 as the biorecognition element. The biorecognition element is immobilized by the affinity between PolyA and the gold electrode and the complementary pairing effect between PolyA and the aptamer, without the need for covalent bonding, effectively reducing preparation costs and operational difficulties.

[0053] This invention utilizes the principle that the specific binding reaction between aptamers and AFB1 leads to a regular change in electrochemical signals to establish the correlation between the response characteristic value (ΔI) of an electrochemical biosensor and the AFB1 content in peanuts, thereby achieving qualitative identification and precise quantitative detection of AFB1. Among these, the PolyA-mediated label-free electrochemical aptamer sensor exhibits excellent linearity in the range of 0.001–1000 nM. R The limit of detection (LOD) is 0.995, and the limit of detection is 0.22 pM. The linear range and sensitivity are significantly improved compared with existing technologies. It also has good specificity and stability, excellent recovery rate in actual sample detection, and does not require expensive instruments and complex pretreatment. It is simple to operate and low in cost, making it more suitable for large-scale and rapid on-site screening of AFB1 residues in peanuts. It can provide technical support for rapid detection of food safety and quality, and is expected to be widely used in the field of food safety testing.

[0054] 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. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications 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 PolyA-mediated label-free electrochemical sensor, characterized in that: A three-electrode system is used as the overall working system, comprising a working electrode, a reference electrode, and an auxiliary electrode. The working electrode is a PolyA-mediated label-free electrochemical aptamer sensor, using the AFB1 aptamer as the biorecognition element. This biorecognition element pairs complementaryly with a DNA fragment containing the PolyA sequence, and is then immobilized on the gold electrode surface by the specific affinity of PolyA for gold. The reference electrode is a silver chloride electrode, and the auxiliary electrode is a platinum wire electrode. The sequences of the AFB1 aptamer include: 5′-GGG TGG GTG GGT GGG GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTAGGC CCA CA-3′ and 5′-ACA CCC GGA TCG CTT CCC GTG CTC TGT GTC TCT CTG TTG TGCACG GGT TG GGG TGG GTG GGT GGG-3′. The sequence of PolyA is: 5′-TGT GGG CCT AGC. GAAGGG CAC GAG ACA CAG AGA GAC AAC ACG TGC CCA AC TTT TTT A10 TTT TTT CAA CCCGTG CAC AAC AGA GAG ACA CAG AGC ACG GGA AGC GAT CCG GGT GT-3′.

2. A method for preparing a PolyA-mediated label-free electrochemical sensor as described in claim 1, comprising the following steps: (1) Pretreatment of working electrode: Using Au electrode as base electrode, the electrode is polished with alumina slurry, ultrasonically cleaned with anhydrous ethanol and first-grade water, electrochemically cleaned with 0.6 M H2SO4 solution, rinsed with deionized water and dried with nitrogen gas flow, and then set aside for use. (2) AFB1 aptamer folds to form G-quadruplex: AFB1 aptamer was prepared as a 150 μM stock solution, and 10 mM K was added. + The folding buffer was diluted to a final concentration of 6 μM, and then subjected to heat denaturation at 95 °C for 5 min and annealing at 25 °C for 4 h for later use. (3) Sensor construction: S1. Drop 1.0 μM, 10 μL PolyA solution onto the pretreated Au electrode surface, incubate overnight at 25 °C, rinse with TE buffer, and dry with nitrogen gas flow; S2. The 10 μM, 10 μLAFB1 aptamer G-tetrachain solution prepared in step (2) was incubated at 25 °C for 60 min, rinsed with TE buffer, and dried with nitrogen gas. S3. Drop 10 μL of 0.5 mM mercaptohexanol (MCH) solution onto the substrate, incubate at 25 °C for 45 min, rinse with TE buffer, and dry with nitrogen gas. S4. Incubate in 10 μL of 800 μM methylene blue (MB) solution for 80 min, rinse with TE buffer, and dry with nitrogen gas to obtain the product; The TE buffer formulation is as follows: TE buffer contains 50 mM Tris-HCl, 1.0 mM EDTA, pH=7.4; K + The folding buffer contains 50 mM Tris-HCl, 100 mM KCl, and 0.1 mM EDTA, with a pH of 7.

4.

3. The method for preparing the PolyA-mediated label-free electrochemical sensor according to claim 2, characterized in that: In step (1), polishing is performed sequentially using 0.2 μm and 0.05 μm alumina slurries, ultrasonic cleaning is performed for 5 min each time, and electrochemical cleaning is performed until a stable cyclic voltammogram is obtained.

4. The method for preparing the PolyA-mediated label-free electrochemical sensor according to claim 3, characterized in that: In step (1), the alumina slurry polishing is specifically as follows: place the gold electrode on a polishing cloth, add a small amount of alumina slurry, and gently rotate the electrode by hand to polish the electrode surface evenly until the electrode surface presents a uniform mirror gloss, without scratches or stains; after each polishing, immediately rinse the electrode surface with deionized water; the ultrasonic cleaning is specifically as follows: place the polished gold electrode into anhydrous ethanol and deionized water in sequence for ultrasonic cleaning, with the ultrasonic power controlled at 200 W; after ultrasonic cleaning, remove the electrode and rinse it repeatedly with deionized water 3-5 times to ensure that there are no residual reagents on the electrode surface; the electrochemical cleaning is specifically as follows: use the ultrasonically cleaned gold electrode as the working electrode, the silver chloride electrode as the reference electrode, and the platinum wire electrode as the auxiliary electrode to form a three-electrode system, and place it in a 0.6 M H2SO4 solution; use cyclic voltammetry (CV) to perform electrochemical cleaning on the gold electrode, with the scanning range set to -0.2 V to 1.5 V and the scanning rate at 50 Hz. mV / s ensures that the electrode surface is clean, free of impurities, and has uniform electrochemical performance. Then, rinse the electrochemically cleaned gold electrode with plenty of deionized water 3-5 times, and slowly blow nitrogen gas onto the electrode surface until the electrode is completely dry. Place it in a desiccator for later use.

5. The method for preparing a PolyA-mediated label-free electrochemical sensor according to claim 2, characterized in that: The specific operation in step (3) is as follows: S1. PolyA Immobilization: Take the pretreated and dried gold electrode, accurately pipette 10 μL of 1.0 μM PolyA solution, and uniformly drop it onto the working surface of the gold electrode; incubate the electrode overnight at 25 ℃. Utilizing the specific affinity of PolyA molecules for the gold electrode surface, PolyA is firmly immobilized on the gold electrode surface, forming a stable PolyA-modified layer; after incubation, gently rinse the electrode surface three times with TE buffer, each rinse lasting 10 s, using gentle pressure to avoid damaging the PolyA-modified layer, thus removing loosely bound and not firmly immobilized PolyA from the electrode surface; then slowly purge the electrode surface with nitrogen gas until completely dry, obtaining the PolyA-modified gold electrode. S2. Aptamer binding: First, prepare a 150 μM stock solution of AFB1 aptamer, then add 10 mM K. + Folding buffer was used to dilute and bring the aptamer concentration to a final level of 6 μM. The aptamer was then subjected to heat denaturation at 95 °C for 5 min and annealing at 25 °C for 4 h to fold into a G-quadruplex with specific AFB1 recognition capability. Using a pipette, 10 μL of the folded AFB1 aptamer solution was evenly applied to the electrode surface of the modified electrode (S1) and incubated at 25 °C for 60 min. This allowed the aptamer to firmly bind to the PolyA-modified electrode surface through complementary pairing with the PolyA sequence. After incubation, the electrode surface was rinsed three times with TE buffer to remove uncomplementary free aptamers. The electrode was then purged with nitrogen and dried to obtain the aptamer-modified electrode. S3. Non-specific site blocking: Take the aptamer-modified electrode obtained in S2, use a pipette to draw 10 μL of 0.5 mM MCH solution, and evenly drop it onto the electrode surface. Incubate at 25 ℃ for 45 min. The thiol groups in the MCH molecule bind to the residual active sites of unbound PolyA on the gold electrode surface, blocking the non-specific binding sites on the electrode surface. This effectively avoids the non-specific adsorption of aptamers, AFB1 and signal molecules, and reduces background interference. After incubation, the electrode surface was rinsed three times with TE buffer to remove unbound free MCH, and then dried by nitrogen purging to obtain the sealed modified electrode. S4. MB Incubation: Take the modified electrode blocked by MCH in S3, pipette 10 μL of 800 μM MB solution, and evenly drop it onto the electrode surface. Incubate at 25 ℃ for 80 min to allow MB molecules to specifically bind to the aptamer G-quadruplex, serving as an electrochemical response signal molecule. After incubation, rinse the electrode surface three times with TE buffer to remove unbound free MB, and slowly purge and dry with nitrogen to finally prepare the PolyA-mediated label-free electrochemical sensor. Store the prepared sensor in a sealed container at 4℃ for up to 10 days.

6. An application of the PolyA-mediated label-free electrochemical sensor for AFB1 detection as described in claim 1.

7. The AFB1 detection application according to claim 6, characterized in that: The steps of the AFB1 detection application include incubating the sensor with a peanut sample solution, detecting the electrochemical response signal using square wave stripping voltammetry (SWV), and using the peak current difference ΔI to reflect the AFB1 concentration, thereby achieving quantitative detection of AFB1.

8. The AFB1 detection application according to claim 7, characterized in that, The detected AFB1 concentration range is 0.001~1000 nM, the linear correlation coefficient R²≥0.995, and the limit of detection is 0.22 pM.

9. The AFB1 detection application according to claim 8, characterized in that: The peanut samples were treated using a five-fold matrix dilution method, and incubated at a constant temperature of 37 ℃ for 60 min. The SWV detection parameters were a scan range of -0.5 V to 0.5 V and a scan rate of 25 mV / s.

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