A sensor for detecting aflatoxin b1 and a preparation method thereof

Abstract

This invention provides a sensor and its preparation method for detecting aflatoxin B1, belonging to the fields of analytical chemistry and food safety detection technology. The sensor includes a fluorescence quenching unit and a molecular recognition and signaling unit. The fluorescence quenching unit is an amino-functionalized zirconium-based metal-organic framework material UiO-66-NH2. The molecular recognition and signaling unit is a specific nucleic acid aptamer for aflatoxin B1, one end of which is covalently modified with a fluorescent dye. The nucleic acid aptamer binds to the surface of UiO-66-NH2 through adsorption. In the absence of aflatoxin B1, the fluorescence is quenched; in the presence of aflatoxin B1, the nucleic acid aptamer binds to the target analyte and dissociates from the UiO-66-NH2 surface, restoring fluorescence. This invention constructs a fluorescence "turn-on" sensor based on the UiO-66-NH2 / TAMRA aptamer, possessing both high sensitivity and specificity. Through visualization hydrogel technology, it achieves low-cost, rapid, and on-site detection of aflatoxin B1 in complex matrices.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry and food safety testing technology, and more specifically, to a sensor for detecting aflatoxin B1 and a method for its preparation. Background Technology

[0002] Aflatoxin B1 (AFB1) is one of the most potent known chemical carcinogens, classified as a Group 1 carcinogen, posing a serious threat to human health. Therefore, establishing rapid and accurate AFB1 detection methods is crucial for food safety monitoring.

[0003] Currently, the mainstream detection methods for AFB1 mainly include chromatography (such as HPLC-MS) and immunoassay (such as ELISA). While chromatography offers high precision and accuracy, it typically requires expensive equipment, specialized operators, and cumbersome pretreatment steps, making it difficult to meet the needs of rapid on-site screening. Immunoassay, although relatively quick, suffers from issues such as poor stability of its core reagent antibodies, susceptibility to interference from complex matrices, significant batch-to-batch variability, and high cost, limiting its widespread application in primary care and field testing.

[0004] Aptamers are single-stranded DNA or RNA molecules obtained through in vitro screening that can specifically bind to target molecules. Compared with antibodies, aptamers have significant advantages such as good stability, ease of chemical synthesis and modification, and low cost. Aptamer-based "turn-on" fluorescent sensors have attracted much attention due to their flexible design and ease of operation. Their working principle typically involves binding a fluorescently labeled aptamer to a quenching material. When the target analyte is absent, the fluorescence is quenched ("Off" state); when the target analyte is present, the aptamer binds to it and undergoes a conformational change, thereby detaching from the quenching material and restoring the fluorescence ("On" state), achieving "on" detection of the signal.

[0005] High-performance quenching materials are crucial for constructing such sensors. Traditional quenching materials, such as graphene oxide, often suffer from drawbacks such as complex preparation processes and insufficient stability. Metal-organic frameworks (MOFs), a class of porous crystalline materials formed by the self-assembly of metal ions and organic ligands, possess advantages such as large specific surface area, tunable structure, and good stability, showing great potential as novel fluorescence quenchers. In particular, zirconium-based MOFs (such as the UiO-66 series) are ideally suited for detection in complex food matrices due to their excellent chemical and hydrothermal stability.

[0006] However, there are currently no reports on using amino-functionalized UiO-66-NH2 as a quencher, combined with fluorescently labeled AFB1 aptamers, to construct a rapid, visual "turn-on" fluorescent sensor for detecting AFB1. In particular, further integration into thermosensitive hydrogels to develop an immobilized sensing platform suitable for field use remains a promising research direction with significant application value.

[0007] Therefore, in view of the above situation, there is an urgent need to develop a sensor and preparation method for detecting aflatoxin B1 in order to overcome the shortcomings in current practical applications. Summary of the Invention

[0008] The purpose of this invention is to provide a sensor for detecting aflatoxin B1 and a method for preparing it, in order to solve the problems mentioned in the background art.

[0009] This invention is implemented as follows: a sensor for detecting aflatoxin B1, comprising: The fluorescence quenching unit is composed of a zirconium-based metal-organic framework material, wherein the organic ligand of the zirconium-based metal-organic framework material contains an amino group (such as an aromatic carboxylic acid containing an amino group). The molecular recognition and signaling unit is composed of a specific nucleic acid aptamer for aflatoxin B1, and one end of the nucleic acid aptamer is covalently modified with a fluorescent dye. The nucleic acid aptamer binds to the surface of the zirconium-based metal-organic framework material through adsorption. In the absence of the target substance, the fluorescence is quenched. When aflatoxin B1 is present, the nucleic acid aptamer binds to the target substance and dissociates from the surface of the zirconium-based metal-organic framework material, resulting in the recovery of fluorescence.

[0010] Optionally, the organic ligand is 2-aminoterephthalic acid, and the zirconium-based metal-organic framework material is UiO-66-NH2.

[0011] Optionally, the fluorescent dye is selected from any one of tetramethylrhodamine, carboxyfluorescein, or Cy3.

[0012] Optionally, the nucleotide sequence of the aflatoxin B1-specific nucleic acid aptamer is shown in SEQ ID NO:1: 5'-TCATCTATCTATGGTACATTACTATCTGTAATGTGATAT-3'.

[0013] Optionally, the concentration of the nucleic acid aptamer is 100 nM, and the concentration of the zirconium-based metal-organic framework material is 26 µM.

[0014] Optionally, the buffer system of the sensor is phosphate-buffered saline (PBS) at pH 7.5.

[0015] Optionally, it also includes a carrier unit, which is a hydrogel, in which the zirconium-based metal-organic framework material and the nucleic acid aptamer are physically embedded.

[0016] Optionally, the hydrogel is a thermosensitive hydrogel, which is selected from any one of poloxamer 407, poloxamer 188, or poly-N-isopropylacrylamide.

[0017] The working principle of the sensor of this invention is as follows: In the absence of the target analyte aflatoxin B1, a negatively charged fluorescently labeled aptamer is adsorbed onto the surface of UiO-66-NH2 through electrostatic interactions and van der Waals forces. At this time, the fluorescent dye on the aptamer is extremely close to the metal-organic framework material, resulting in photoinduced electron transfer. This leads to the efficient quenching of the emitted light from the fluorescent dye, and the sensing system is in a fluorescence-off state. When the target analyte aflatoxin B1 is present in the system, the aptamer, with its high specificity and affinity, binds to aflatoxin B1, inducing a conformational change and forming a stable complex (such as a G-quadruplex or a hairpin structure), thereby dissociating from the surface of the metal-organic framework material. This process significantly increases the physical distance between the fluorescent dye and the metal-organic framework material quencher, blocking the electron transfer path, allowing the fluorescence signal to recover, and the system switches to a fluorescence-on state. Furthermore, the fluorescence recovery intensity of the system is positively correlated with the concentration of aflatoxin B1 in the solution within a certain range, thereby enabling highly sensitive quantitative detection of aflatoxin B1.

[0018] Another object of the present invention is a method for preparing a sensor for detecting aflatoxin B1, comprising the following steps: Step 1: Synthesis of zirconium-based metal-organic framework materials: Zirconium salt and 2-aminoterephthalic acid were dissolved in an organic solvent, a structure modifier was added, and after hydrothermal reaction, the product was washed and dried to obtain UiO-66-NH2 powder. Step 2: Preparation of the sensor working fluid: The UiO-66-NH2 powder obtained in step one is dispersed in a buffer solution to obtain an MOF dispersion; the fluorescently labeled aflatoxin B1 aptamer is dissolved in the buffer solution to obtain an aptamer solution; the MOF dispersion and the aptamer solution are mixed and incubated to allow the aptamer to adsorb onto the surface of UiO-66-NH2, thus obtaining the sensor working solution.

[0019] Optionally, in step one, the zirconium salt is zirconium tetrachloride, the organic solvent is N,N-dimethylformamide (DMF), and the structure modifier is acetic acid; the hydrothermal reaction conditions are: temperature 120°C, reaction time 24 hours.

[0020] Optionally, in step two, the buffer solution is a phosphate buffer solution with a pH of 7.5; the incubation temperature is room temperature to 37°C, and the incubation time is 60 minutes.

[0021] Another object of the present invention is a method for preparing an immobilized sensing membrane based on the sensor working fluid, comprising the following steps: Thermosensitive hydrogel polymers are dissolved in low-temperature ultrapure water and stirred until completely dissolved to obtain a transparent sol; The sensor working solution obtained in step two of the preparation method of the sensor for detecting aflatoxin B1 is mixed evenly with the transparent sol at low temperature; The mixture is placed at room temperature or higher, and the sol-gel transition characteristics of the thermosensitive hydrogel are utilized to solidify it into a non-flowing hydrogel, thus obtaining the immobilized sensing membrane.

[0022] Optionally, the thermosensitive hydrogel polymer is poloxamer 407; the mass ratio of poloxamer 407 to ultrapure water is 1:4.

[0023] Another object of the present invention is a method for detecting aflatoxin B1, using the sensor described above or the sensor working solution prepared by the method, the method comprising the following steps: Step 1: Mixed Incubation The sensor working solution is mixed with the sample solution to be tested and incubated under set conditions; Step 2, Signal Detection: Detect the fluorescence signal intensity of the mixture after incubation; Step 3: Quantitative Analysis Based on the quantitative relationship between the fluorescence signal intensity and the aflatoxin B1 concentration, the content of aflatoxin B1 in the sample to be tested is determined.

[0024] Optionally, the set conditions include a phosphate buffer system with pH 7.5, an incubation temperature of 24-37°C, and an incubation time of 60-80 minutes; the excitation wavelength for fluorescence detection is 252 nm, and the emission wavelength is 586 nm.

[0025] Optionally, the detection method further includes visual semi-quantitative detection based on hydrogel carriers, specifically including: The sample solution to be tested is dropped onto a hydrogel carrier loaded with the working solution of the sensor, and the change in fluorescence color is observed under 365nm ultraviolet light. The concentration range of aflatoxin B1 in the sample to be tested is estimated by comparing it with a standard colorimetric card or standard fluorescence scale.

[0026] Another object of the present invention is a kit for the detection of aflatoxin B1, comprising: Core detection component: the sensor described above, or the sensor working solution prepared by the method described above; Auxiliary reagents: buffer solutions used to dissolve or dilute samples.

[0027] Optionally, it also includes an aflatoxin B1 standard solution, which is used to plot a standard curve or as a positive control.

[0028] Optionally, it further includes a thermosensitive hydrogel polymer, which is used to mix with the sensor working fluid to prepare an immobilized sensing membrane.

[0029] Another object of the present invention is the application of the sensor, the preparation method, the detection method, or the kit described herein in the detection of aflatoxin B1.

[0030] Optionally, the object being tested is selected from food, feed, or agricultural products.

[0031] Optionally, the food, feed, or agricultural product includes grains, grain products, fruit and vegetable juices, or fermented liquids.

[0032] The present invention provides a sensor for detecting aflatoxin B1 and a method for its preparation, which has the following beneficial effects: This invention has significant advantages such as high sensitivity, high selectivity, simple operation, low cost and strong practicality.

[0033] Specifically, this is reflected in the following aspects: The efficient quenching of TAMRA fluorescence by UiO-66-NH2 and the specific recognition of aflatoxin B1 by the aptamer achieve a "turn-on" signal response with a detection limit as low as 0.012µM; the sensor exhibits high specificity for aflatoxin B1 and strong anti-interference ability, ensuring accuracy in complex matrices; the detection process is accompanied by obvious fluorescence color changes, and combined with the hydrogel carrier, it enables rapid on-site screening without complex instruments; the materials used are low-cost and biocompatible, conforming to the concept of green chemistry; and it has been successfully applied to the detection of various actual agricultural and food samples, with results consistent with national standard methods, demonstrating its reliability and wide applicability.

[0034] Other features and advantages of the invention will become clear from the following detailed description of exemplary embodiments of the invention with reference to the accompanying drawings. Attached Figure Description

[0035] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0036] Figure 1This is a schematic diagram illustrating the principle of the present invention for detecting aflatoxin B1 based on a fluorescent aptamer probe; Figure 2 The Fourier transform infrared spectra of the key components (AFB1, TAMRA-labeled aptamer, UiO-66-NH2, composite probe and binding product) in the examples are shown below. Figure 3 The above-mentioned examples in the embodiments ( Figure 2 UV-Vis absorption spectra of each component; Figure 4 This is a comparison of the fluorescence lifetime decay curves of TAMRA-Apt, MOF-TAMRA-Apt, and MOF-TAMRA-Apt-AFB1 in the examples. Figure 5 The curve showing the effect of UiO-66-NH2 concentration on the fluorescence quenching efficiency of TAMRA-Apt in the examples; Figure 6 This is a graph showing the effect of different pH values ​​on the detection of AFB1 fluorescence recovery signal by the sensor in the examples; Figure 7 The following is a kinetic curve illustrating the effect of different reaction times on the sensor's detection of AFB1 fluorescence recovery signal in the examples; Figure 8 The following are examples of fluorescence response spectra of the sensor to different concentrations of AFB1 (A) and the corresponding fluorescence intensity ratio (F / F0) versus the standard working curve of AFB1 concentration (B). Figure 9 The bar chart shows the test results of the sensor's selectivity (A) and anti-interference (B) against AFB1 and other potential interfering substances in the embodiment. Figure 10 These are images showing the fluorescence response of the hydrogel sensing membrane of the load sensor in the embodiment at different concentrations of AFB1. Figure 11 This is a schematic diagram and corresponding visual image of the detection results of AFB1 on an actual grain sample using a hydrogel sensing membrane, as shown in the example. Detailed Implementation

[0037] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0038] Example 1: Preparation of metal-organic framework UiO-66-NH2 Accurately weigh 0.36 g of 2-aminoterephthalic acid and 0.46 g of zirconium tetrachloride (ZrCl4) and place them in a 50 mL beaker. Add 25 mL of N,N-dimethylformamide to the beaker and stir magnetically until the solid is substantially dissolved. Then, add 2.0 mL of glacial acetic acid to the solution and continue stirring until homogeneous. Sonicate the resulting mixture for 2 hours to ensure thorough dispersion of the reactants. Then, transfer the mixture to a 100 mL high-pressure reactor lined with polytetrafluoroethylene, seal it, and place it in an oven at 120 °C for 24 hours.

[0039] After the reaction was complete, the reactor was allowed to cool naturally to room temperature. The resulting suspension was centrifuged at 8000 rpm for 10 minutes, and the precipitate was collected. The precipitate was washed three times, successively with N,N-dimethylformamide and anhydrous ethanol, to thoroughly remove unreacted raw materials and residual solvent molecules. Finally, the resulting light yellow solid product was placed in a vacuum drying oven and dried overnight at 70°C. The dried product was ground to obtain UiO-66-NH2 powder, which was then stored in a desiccator for later use.

[0040] Example 2: Preparation and Condition Optimization of Working Solution for Fluorescent Aptamer Sensor This embodiment describes in detail the preparation process of the core working fluid of the sensor and systematically explores the effects of material concentration and environmental factors on detection performance in order to determine the optimal experimental conditions.

[0041] 1) Preparation of reagents and solutions Phosphate buffer: Prepare a 10mM PBS solution with pH 7.5 as the basic buffer system for subsequent experiments.

[0042] MOF dispersion: Accurately weigh 1.0 mg of UiO-66-NH2 powder prepared in Example 1 and add it to 1.0 mL of PBS buffer. Disperse by sonication for 30 minutes to ensure thorough and uniform dispersion, preparing a stock solution with a concentration of 1 mg / mL. Store at 4°C for later use. Before use, dilute with PBS to the required concentration according to experimental needs.

[0043] TAMRA-Apt solution: Dissolve the synthesized 5'-TAMRA-labeled aflatoxin B1 aptamer lyophilized powder in an appropriate amount of PBS buffer to prepare a 100µM stock solution. Aliquot the solution and store it at -20℃ protected from light, avoiding repeated freeze-thaw cycles.

[0044] 2) Optimization of MOF concentration To determine the optimal amount of fluorescence quencher, 2 mL of PBS buffer and 10 µL of LTAMRA-Apt stock solution were added to a quartz cuvette to bring the final concentration of the aptamer to 100 nM, and the mixture was incubated at 37 °C for 60 minutes.

[0045] Subsequently, different volumes of MOF dispersion were added to establish final MOF concentration gradients of 0, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32 µM. After thorough mixing, the mixture was incubated for another 60 minutes.

[0046] Detection was performed using a fluorescence spectrometer. The excitation wavelength was set to 252 nm, and the emission spectrum was scanned, with the fluorescence intensity F at 586 nm recorded. The fluorescence intensity of the aptamer without MOF was taken as F0, and the fluorescence intensity ratio (F / F0) at different MOF concentrations was calculated. Figure 5 As shown, the fluorescence intensity gradually decreases with increasing MOF concentration. When the MOF concentration reaches 26 µM, the fluorescence quenching efficiency ((F0-F) / F0) exceeds 90% and the curve tends to flatten, indicating that the quenching effect has reached saturation. Therefore, 26 µM was selected as the optimal working concentration of MOF in subsequent experiments.

[0047] 3) Optimization of detection conditions pH Optimization: The effect of solution pH on sensor performance was investigated. Sensor working solutions (containing 100 nM TAMRA-Apt and 26 µM MOF) were prepared using 10 mM PBS buffer at different pH values ​​(6.5, 7.0, 7.5, 8.0, 8.5, 9.0). The same concentration of aflatoxin B1 (e.g., 10 µM) was added to each working solution, and after incubation for 70 minutes, the fluorescence recovery intensity at 586 nm was measured. Experimental results (e.g.) Figure 6 As shown in the figure, the sensor exhibits the strongest fluorescence recovery signal and the best stability at pH 7.5, therefore pH 7.5 is selected as the optimal detection environment.

[0048] Incubation time optimization: Under optimal pH conditions, aflatoxin B1 (10 µM) was added to the sensor working solution, and the effect of reaction time on fluorescence signal was investigated. Fluorescence intensity was measured at 0, 10, 20, 30, 40, 50, 60, 70, and 80 minutes of reaction. Figure 7 As shown, the fluorescence intensity increases with time, reaching a plateau after approximately 70 minutes and then ceasing to change significantly. Therefore, the optimal incubation time was determined to be 70 minutes.

[0049] Temperature optimization: Aflatoxin B1 detection experiments were conducted at 15℃, 20℃, 24℃, 30℃, 37℃, and 45℃. Results showed that the sensor performance was stable within the range of room temperature to physiological temperature (24-37℃), exhibiting strong fluorescence response and good reproducibility. For ease of operation, subsequent experiments can be conducted at room temperature (approximately 24-25℃) or 37℃.

[0050] Example 3: Quantitative Detection and Standard Curve of Aflatoxin B1 This embodiment details a method for the quantitative detection of aflatoxin B1 using a constructed fluorescent aptamer sensor under optimal experimental conditions, and establishes a standard working curve. The specific detection steps are as follows: First, a basic sensing system was constructed. 2 mL of PBS buffer (10 mM, pH 7.5) and 10 µL of TAMRA-Apt solution were added to a series of quartz cuvettes, respectively, to bring the final aptamer concentration to 100 nM. The cuvettes were incubated at 37°C for 60 minutes. Subsequently, 26 µL of MOF dispersion (final concentration 26 µM) was added to each cuvette, and incubation continued at room temperature for another 60 minutes. At this point, the fluorescence of TAMRA was efficiently quenched by UiO-66-NH2, and the system was in a "closed" state. The fluorescence intensity at this point was recorded as the background value F0.

[0051] Next, a standard curve for aflatoxin B1 was determined. Different volumes of aflatoxin B1 standard solution were added dropwise to the quenched system to construct a concentration gradient, resulting in final concentrations of 0, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 µM of aflatoxin B1. After thorough mixing, the mixture was reacted at room temperature for 70 minutes to allow aflatoxin B1 to fully bind to the aptamer and induce fluorescence recovery. The fluorescence intensity F (excitation wavelength 252 nm, emission wavelength 586 nm) of each solution was measured using a fluorescence spectrometer. Three parallel experiments were performed for each concentration point to ensure data reliability.

[0052] Data analysis and results: such as Figure 8 As shown, a standard curve was plotted with the fluorescence intensity ratio (F / F0) as the ordinate (y) and the AFB1 concentration as the abscissa (x). Figure 8 B). Experimental results show that within the concentration range of 0-20 µM, the F / F0 value exhibits a good linear relationship with the AFB1 concentration. The linear regression equation is y = 0.648 + 0.175x, with a correlation coefficient R² = 0.993. Based on a signal-to-noise ratio of 3 (S / N = 3), the limit of detection (LOD) for AFB1 using this method is as low as 0.012 µM, demonstrating extremely high detection sensitivity.

[0053] Example 4: Evaluation of Sensor Selectivity and Interference Immunity This embodiment aims to evaluate the specificity of the constructed fluorescent aptamer sensor for aflatoxin B1 recognition and its anti-interference ability under complex environments. Specifically: 1) Selective Experiment To examine the sensor's specific recognition capability, different potential interfering substances, each at a concentration of 4 µM, were added to the sensor's working solution under the same experimental conditions. The test targets included: other fungal toxins (deoxynivalenol (DON), ochratoxin (AOTA), and common metal ions (K+). + The reaction mixture contained Mg²⁺, Zn²⁺, and Fe²⁺ (added in chloride form), common anions (Br⁻, F⁻), and small molecule organic compounds (vitamin B, vitamin C, L-alanine). Aflatoxin B1 (4 µM) was used as a positive control. After the reaction, the fluorescence intensity of each system was measured at 586 nm, and the fluorescence intensity ratio (F / F0) was calculated.

[0054] Experimental results are as follows Figure 9 As shown in Figure A, after adding the same concentration of each interfering substance, the fluorescence intensity of the system did not change significantly, and the F / F0 value remained at a low level. In contrast, a significant fluorescence recovery signal was observed only in the group containing aflatoxin B1. This result indicates that the sensor can accurately identify aflatoxin B1 and has high selectivity for other structural analogs and common coexisting ions.

[0055] 2) Anti-interference test To further verify the sensor's applicability in complex matrices, anti-interference performance tests were conducted. Aflatoxin B1 (final concentration 2 µM) and a mixture of all the aforementioned interfering substances (each at a concentration of 4 µM) were added to the sensor's working solution to simulate the complex coexistence environment that may exist in actual samples. The fluorescence signal of the mixed system after the reaction was measured and compared with the fluorescence response of adding 2 µM aflatoxin B1 alone.

[0056] like Figure 9 As shown in Figure B, even with a large number of interfering substances present, the fluorescence response intensity of the sensor to aflatoxin B1 is basically consistent with that when detecting aflatoxin B1 alone, without significant inhibition or enhancement. This fully demonstrates that the sensor has excellent anti-interference performance, can accurately detect aflatoxin B1 in complex backgrounds, and has good potential for practical applications.

[0057] Example 5: Preparation and Visual Detection of Hydrogel Sensing Membranes This embodiment demonstrates the process of immobilizing liquid fluorescent probes to construct a portable sensing membrane and verifies its visualization detection capability without instrument assistance.

[0058] 1) Preparation of hydrogel sensing membranes First, prepare the thermosensitive matrix. Weigh 1.0 g of poloxamer 407 powder, add 4.0 g of ultrapure water, and place in a refrigerator at 4°C overnight. During this period, stir intermittently until the powder is completely dissolved, forming a uniform, transparent, viscous sol.

[0059] Subsequently, a composite sensing system was constructed. 1 mL of the sensor working solution (containing 100 nM TAMRA-Apt and 26 µM MOF) prepared according to the method in Example 2 was taken and thoroughly mixed with 1 mL of the pre-cooled poloxamer sol at 4°C. The mixture was then dropped onto a black 96-well plate or a clean glass slide and placed at room temperature (>20°C). Utilizing the thermotropic gelation properties of poloxamer, the sol underwent a phase transition within minutes, transforming into a non-flowing, transparent hydrogel membrane, thereby obtaining a fixed sensing membrane.

[0060] 2) Visualized detection 10 µL of aflatoxin B1 standard solutions of different concentrations (0, 1, 5, 10, and 20 µM) were each dropped onto the surface of the prepared hydrogel membrane. The membrane was reacted at room temperature for 70 minutes to allow the aflatoxin B1 to fully contact the probe inside the membrane and induce fluorescence recovery.

[0061] During the detection, the sample was placed in a dark room and irradiated with a handheld ultraviolet lamp at a wavelength of 365nm. A fluorescence photograph was then taken using a digital camera (e.g., Figure 10 (As shown in the image). Observation results show that with the increase of aflatoxin B1 concentration, the intensity of the orange-red fluorescence emitted by the hydrogel membrane exhibits a significant gradient increase, with the color gradually changing from almost colorless to bright orange-red. This result indicates that the hydrogel-based immobilized sensing platform can achieve rapid semi-quantitative screening of aflatoxin B1 without relying on sophisticated instruments, simply by observing changes in fluorescence color with the naked eye, demonstrating good portability and potential for field application.

[0062] Example 6: Actual Sample Testing This embodiment aims to verify the applicability and accuracy of the constructed sensor in complex real-world samples. Grains (corn, wheat, millet, soybeans) and fruit juices (apple juice, tomato juice) were selected as representative matrices for the experiment, and sample pretreatment, spike recovery experiments, and quantitative and visual detection were performed.

[0063] First, sample pretreatment was performed to extract the test solution. For grain samples, the grains were pulverized and passed through a 60-mesh sieve. 1.0 g of powder was accurately weighed and placed in a 15 mL centrifuge tube. 5 mL of extraction solvent (acetonitrile:water = 84:16, v / v) was added. After vortexing for 10 minutes and ultrasonic extraction for 10 minutes, the mixture was centrifuged at 4000 rpm for 10 minutes. 1.0 mL of the supernatant was transferred to another centrifuge tube and dried under nitrogen at 50°C. The residue was then reconstituted with 1.0 mL of PBS buffer (10 mM, pH 7.5) and filtered through a 0.22 µm microporous membrane to obtain the grain sample test solution. For fruit juice samples, 1.0 mL of clarified fruit juice was added to 1.0 mL of ethyl acetate, vortexed for 2 minutes, and centrifuged at 4000 rpm for 5 minutes. The upper organic phase was collected and dried under nitrogen. The mixture was then reconstituted with 1.0 mL of PBS buffer and filtered to obtain the fruit juice sample test solution.

[0064] In the spiked recovery experiment and accuracy verification stage, blank samples with aflatoxin B1 levels below the detection limit or undetectable by preliminary HPLC determination were selected and processed to obtain blank matrix solutions according to the above method. Aflatoxin B1 standards at low, medium, and high concentration levels (equivalent to 1, 10, and 20 µM in the sample) were added to the blank matrix solutions, with three parallel samples prepared for each level to evaluate the precision of the experiment.

[0065] The quantitative detection steps are as follows: Take 100 µL of the spiked sample solution and mix it with 900 µL of PBS buffer and 10 µL of LTMRA-Apt stock solution to make the total volume approximately 1 mL and maintain the final aptamer concentration at approximately 100 nM. Subsequently, MOF is added according to the steps described in Example 3, and the fluorescence intensity is measured. The actual content and recovery rate of aflatoxin B1 in the sample are calculated based on the standard curve.

[0066] Visualization was also performed to assess its potential for field application. 10 µL of the spiked sample solution was directly added to the hydrogel sensing membrane prepared in Example 5 and incubated under the same conditions. Subsequently, the membrane was observed under UV light and fluorescence images were taken. Figure 11 By comparing the fluorescence intensity with a series of standard concentrations, a semi-quantitative estimation of the aflatoxin B1 content in the sample can be achieved.

[0067] also, Figure 1 This is a schematic diagram illustrating the principle of the present invention for detecting aflatoxin B1 based on a fluorescent aptamer probe, demonstrating the "turn-on" detection mechanism and the application of hydrogel visualization. Figure 2 The Fourier transform infrared spectra of each key component (aflatoxin B1, TAMRA-labeled aptamer, UiO-66-NH2, composite probe and binding product) in the examples are used to verify the successful preparation and interaction of the materials. Figure 3 The above-mentioned examples in the embodiments ( Figure 2 The UV-Vis absorption spectra of each component further corroborate the assembly process of the nanoprobe. Figure 4 The comparison diagram of fluorescence lifetime decay curves of TAMRA-Apt, MOF-TAMRA-Apt and MOF-TAMRA-Apt-AFB1 in the examples reveals the occurrence of fluorescence resonance energy transfer or static quenching.

[0068] The above embodiments of the present invention provide a sensor and its preparation method for detecting aflatoxin B1, which is a fluorescent nanoprobe based on a metal-organic framework (UiO-66-NH2) and aptamer-labeled dye (TAMRA) for detecting aflatoxin B1. Compared with the prior art, the main advantages are as follows: 1) Utilizing the superior quenching efficiency of UiO-66-NH2 for TAMRA (tetramethylrhodamine) fluorescence, combined with the high affinity of the aptamer for aflatoxin B1, a significant "turn-on" signal response was achieved. This method achieves a detection limit for aflatoxin B1 as low as 0.012 µM, meeting the requirements for detecting trace amounts of aflatoxin B1 in food.

[0069] 2) The sensor exhibits high specificity for aflatoxin B1. Even when the concentrations of common interfering substances (such as other mycotoxins DON, OTA, metal ions, vitamins, and amino acids) are several times higher than that of aflatoxin B1, it produces almost no interfering signals, ensuring accurate detection in complex food matrices.

[0070] 3) During the detection process, the fluorescence color of the solution changes from colorless to red, resulting in a significant visual effect. Combined with a hydrogel sensing membrane, it enables one-step detection via "sample drop-observation," eliminating the need for complex and expensive instruments. Only a simple UV lamp is required for judgment, making it particularly suitable for rapid on-site screening and portable testing.

[0071] 4) The MOF raw materials used are cheap and readily available, and the aptamers can be chemically synthesized, with costs far lower than those of antibodies; moreover, the materials have good biocompatibility, which is in line with the development direction of green analytical chemistry.

[0072] 5) This method was successfully applied to the detection of aflatoxin B1 in various real samples, including corn, wheat, millet, soybeans, apple juice, and tomato juice. The spiked recoveries ranged from 87.9% to 104.45%, with relative standard deviations of less than 5%, consistent with the results of the national standard HPLC method, demonstrating its reliability and accuracy.

[0073] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A sensor for detecting aflatoxin B1, characterized in that, Includes fluorescence quenching units and molecular recognition and signaling units; The fluorescence quenching unit is an amino-functionalized zirconium-based metal-organic framework material UiO-66-NH2; The molecular recognition and signaling unit is a specific nucleic acid aptamer for aflatoxin B1, and one end of the nucleic acid aptamer is covalently modified with a fluorescent dye. The nucleic acid aptamer binds to the surface of UiO-66-NH2 through adsorption. In the absence of aflatoxin B1, the fluorescence is quenched. In the presence of aflatoxin B1, the nucleic acid aptamer binds to the target and dissociates from the surface of UiO-66-NH2, and the fluorescence is restored.

2. The sensor for detecting aflatoxin B1 according to claim 1, characterized in that, The nucleotide sequence of the aflatoxin B1-specific aptamer is shown in SEQ ID NO:1: 5'-TCATCTATCTATGGTACATTACTATCTGTAATGTGATAT-3'.

3. The sensor for detecting aflatoxin B1 according to claim 1, characterized in that, The fluorescent dye is any one of tetramethylrhodamine, carboxyfluorescein, or Cy3. The final concentration of the nucleic acid aptamer is 100 nM, and the final concentration of UiO-66-NH2 is 26 μM; The sensor's buffer system is a phosphate buffer solution with a pH of 7.

5.

4. The sensor for detecting aflatoxin B1 according to claim 1, characterized in that, It also includes a carrier unit, which is a hydrogel in which UiO-66-NH2 and nucleic acid aptamers are physically embedded; The hydrogel is a thermosensitive hydrogel, selected from any one of poloxamer 407, poloxamer 188, or poly-N-isopropylacrylamide.

5. A method for preparing the working fluid of the sensor as described in any one of claims 1-4, characterized in that, Includes the following steps: Step 1, Synthesis of UiO-66-NH2: Zirconium salt and 2-aminoterephthalic acid are dissolved in an organic solvent, a structure modifier is added, and after hydrothermal reaction, the mixture is washed and dried to obtain UiO-66-NH2 powder; Step 2: Preparation of sensor working solution: Disperse UiO-66-NH2 powder in buffer solution to obtain MOF dispersion, dissolve fluorescently labeled aflatoxin B1 aptamer in buffer solution to obtain aptamer solution, mix MOF dispersion and aptamer solution and incubate to obtain sensor working solution.

6. The method for preparing the working solution according to claim 5, characterized in that, In step one, the zirconium salt is zirconium tetrachloride, the organic solvent is N,N-dimethylformamide, and the structure modifier is acetic acid; the hydrothermal reaction conditions are 120°C for 24 hours. In step two, the buffer solution is a phosphate buffer solution with a pH of 7.5; the incubation temperature is from room temperature to 37°C, and the incubation time is 60 minutes.

7. A method for preparing a sensor working fluid-supported sensing membrane based on the working fluid preparation method described in claim 5, characterized in that, Includes the following steps: Step 1: Dissolve the thermosensitive hydrogel polymer in low-temperature ultrapure water to obtain a transparent sol; Step 2: Mix the sensor working solution with the transparent sol at low temperature until homogeneous; Step 3: Place the mixture at room temperature or higher to solidify it using the sol-gel transition to form a hydrogel, thus obtaining the immobilized sensing membrane. The thermosensitive hydrogel polymer is poloxamer 407, and the mass ratio of poloxamer 407 to ultrapure water is 1:

4.

8. A method for detecting aflatoxin B1, characterized in that, Using the sensor according to any one of claims 1-4, or the sensor working fluid prepared by the method according to any one of claims 5-6, the method comprises the following steps: Step 1: Mix and incubate the sensor working solution with the sample solution to be tested; Step 2: Detect the fluorescence signal intensity of the mixture after incubation; Step 3: Determine the content of aflatoxin B1 in the sample to be tested based on the quantitative relationship between fluorescence signal intensity and aflatoxin B1 concentration; The incubation conditions were: pH 7.5 phosphate buffer system, incubation at 24-37℃ for 60-80 minutes; fluorescence detection excitation wavelength was 252nm, and emission wavelength was 586nm. It also includes visual semi-quantitative detection: the sample solution to be tested is dropped onto the hydrogel carrier of the load sensor, the fluorescence color is observed under 365nm ultraviolet light, and the concentration range is determined by comparing it with the standard color scale.

9. A kit for the detection of aflatoxin B1, characterized in that, Includes core detection components and auxiliary reagents; The core detection component is the sensor according to any one of claims 1-4, or the sensor working solution prepared by the working solution preparation method according to any one of claims 5-6; The auxiliary reagent is a buffer solution used to dissolve or dilute the sample; This also includes aflatoxin B1 standard solution, used to plot a standard curve or as a positive control; It also includes thermosensitive hydrogel polymers for preparing immobilized sensing membranes.

10. The application of the sensor according to any one of claims 1-4, the preparation method according to any one of claims 5-7, the detection method according to claim 8, and the reagent kit according to claim 9, characterized in that, Used to detect aflatoxin B1 in food, feed, or agricultural products; The food, feed, or agricultural products mentioned include grains, grain products, fruit and vegetable juices, or fermented liquids.

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