Eu-mn-hof material, preparation method thereof and application thereof in detection of toxic flavin of burkholderia glumae

Abstract

This invention discloses an Eu-Mn-HOF material, its preparation method, and its application in the detection of flavin in Burkholderia gladioli. This invention utilizes Eu... 3+ Excitation is loaded onto a HOF support to form a red probe. Utilizing the fluorescence internal filtering effect, it becomes a turn-off fluorescent sensor, namely the Eu-Mn-HOF material. This Eu-Mn-HOF material cleverly utilizes Eu... 3+ The signal amplification function of the fluorescence internal filtration effect enables the detection method established using this Eu-Mn-HOF material to achieve an extremely low detection limit (0.839 μM), effectively meeting the needs for accurate detection and early warning of trace toxoflavones. The incubation reaction between the Eu-Mn-HOF material and toxoflavones, as well as the subsequent fluorescence detection process, only requires 15 minutes, and the entire detection cycle can be controlled within 45 minutes. This represents an efficiency improvement of more than 8 times compared to traditional detection methods, greatly satisfying the practical needs of rapid on-site food safety testing.

Description

Technical Field

[0001] This invention belongs to the field of toxoflavone (C7H7N5O2) detection technology, specifically involving an Eu-Mn-HOF material and its preparation method, and its application in the detection of toxoflavone in Burkholderia gladioli. Background Technology

[0002] Toxflavin (TXF) is a water-soluble, small-molecule fatty acid toxin produced by *Burkholderia gladioli* during its growth and reproduction. The high mortality rate of fermented rice noodle poisoning and spoiled tremella poisoning caused by this bacterium is due to the extremely high toxicity of toxflavin, reaching the standards of a potent toxic chemical. As a toxin with severe toxic effects on human and animal cells, toxflavin can disrupt normal cellular physiological functions, leading to organ damage and even failure. Furthermore, toxflavin is highly heat-resistant; ordinary cooking methods such as frying, stir-frying, boiling, and deep-frying cannot destroy it. This means that once food is contaminated with toxflavin, even after cooking, consumption can still cause poisoning. Even more serious is the fact that there is currently no specific antidote for toxflavin poisoning. Once poisoning occurs, patients can only rely on supportive therapy to maintain their lives, posing a significant challenge to the treatment of toxflavin poisoning and highlighting the urgency and importance of developing rapid and accurate detection technologies for toxflavin.

[0003] Currently, the detection of toxoflavones mainly involves instrumental detection, immunoassay, and other methods, but each has its own limitations. Instrumental detection primarily employs spectrophotometry, high-performance liquid chromatography (HPLC), or a combination of both. These methods rely on expensive equipment, have long detection times, require highly skilled operators, and are difficult to implement rapidly on-site. For example, HPLC requires expensive equipment and complex sample pretreatment. Immunoassay methods, lacking reliable toxoflavone haptens, result in excessively high detection limits, failing to accurately detect low levels of toxoflavones in food and frequently producing false negatives or false positives. Furthermore, existing detection methods, when processing complex matrix samples such as fermented rice noodles, require sophisticated enrichment and purification pretreatment, which current methods struggle to effectively remove impurities, leading to inaccurate results. When simultaneously detecting multiple toxins in food (such as bongkrekic acid, isomykrekic acid, and toxoflavones), the significant differences in physicochemical properties among the various toxins make it difficult to balance extraction efficiency and recovery rates using the same extraction and purification methods. Summary of the Invention

[0004] To overcome the shortcomings and drawbacks of existing technologies, the primary objective of this invention is to provide a method for preparing Eu-Mn-HOF materials. This invention utilizes Eu... 3+The excitation load is loaded into the HOF support to form a red probe. Utilizing the fluorescence internal filtration effect, it becomes a turn-off fluorescent sensor, namely the Eu-Mn-HOF material.

[0005] A second objective of this invention is to provide Eu-Mn-HOF materials prepared by the above-described preparation method.

[0006] A third objective of this invention is to provide the application of the aforementioned Eu-Mn-HOF material in the detection of flavin in Burkholderia gladioli. In the specific detection process, Mn... 2+ As an important cofactor of toxoflavone-degrading enzymes, it can bind with toxoflavone to form a fluorescent sensor with targeted recognition, which can effectively meet the needs for accurate detection and early warning of trace toxoflavone.

[0007] The primary objective of this invention is achieved through the following technical solution:

[0008] A method for preparing Eu-Mn-HOF material includes the following steps:

[0009] (1) Synthesis of HOF: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (H3TATB), dimethylformamide (DMF) and deionized water were mixed in a mass-volume ratio of 45~50 mg: 4.5~5.5 mL: 14~16 mL. The solid was collected by centrifugation, washed, soaked, collected by centrifugation again, and dried to prepare HOF.

[0010] (2) Synthesis of Eu-Mn-HOF material: MnCl2·4H2O, Eu(NO3)3·6H2O, ethanol and HOF were prepared in a mass-volume ratio of 10mg:9~11mg:9~11mL:45~55mg. MnCl2·4H2O and Eu(NO3)3·6H2O were dissolved in ethanol and then ultrasonically dispersed in HOF. The mixture was heated, centrifuged, washed and dried to prepare Eu-Mn-HOF material.

[0011] Preferably, the stirring speed in step (1) is 1900~2100 rpm, and the stirring time is 12~24 hours.

[0012] Preferably, the washing in step (1) refers to washing with the organic solvent acetone 3 to 5 times; the soaking refers to soaking with the organic solvent dichloromethane (CH2Cl2) for 3 to 5 days.

[0013] Preferably, the ultrasonic dispersion time in step (2) is 1 to 5 minutes, and the ultrasonic dispersion power is 80 to 120W.

[0014] Preferably, the heating temperature in step (2) is 40~50℃, and the heating time is 1~2 hours.

[0015] Preferably, the washing in step (2) refers to washing with ethanol as an organic solvent 3 to 5 times.

[0016] Preferably, the drying temperature in step (2) is 50~60℃.

[0017] The second objective of this invention is achieved through the following technical solution:

[0018] An Eu-Mn-HOF material was prepared by the above-described preparation method.

[0019] The third objective of this invention is achieved through the following technical solution:

[0020] Application of an Eu-Mn-HOF material in the detection of flavin in Burkholderia gladioli.

[0021] Preferably, the Eu-Mn-HOF material is used to detect Burkholderia gladioli flavin at a wavelength of 280-400 nm.

[0022] Preferably, the Eu-Mn-HOF material is used to detect Burkholderia gladioli flavin at a mass concentration of 0.05~0.1 mg / mL.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] (1) Excellent sensitivity: The Eu-Mn-HOF material described in this invention cleverly utilizes the signal amplification function of the Eu³⁺ fluorescence internal filtering effect. The detection method established by using this fluorescent probe can achieve an extremely low detection limit, up to 0.839 μM, which can effectively meet the needs of accurate detection and early warning of trace toxins.

[0025] (2) Strong anti-interference performance: Eu-Mn-HOF material not only possesses the pore sieving effect of HOF material itself, which can effectively eliminate the interference of macromolecular impurities, but also has a unique interaction mechanism between the Mn²⁺ specific recognition sites modified on its surface and toxoflavones, making the material highly selective for toxoflavones. This dual anti-interference mechanism works synergistically to make the anti-interference rate of this method as high as 95% or more, successfully solving the problem of interference from complex food matrices to the detection of toxoflavones, and greatly improving the accuracy and reliability of the detection results;

[0026] (3) Advantages of rapid detection: In the sample pretreatment stage, by optimizing the extraction method and process, the original cumbersome pretreatment time is greatly shortened to less than 30 minutes; at the same time, the incubation reaction of Eu-Mn-HOF material with toxin and the subsequent fluorescence detection process only take 15 minutes, and the entire detection cycle can be controlled within 45 minutes. Compared with traditional detection methods (which usually take 4-6 hours), the detection efficiency is increased by more than 8 times, which greatly meets the actual needs of rapid on-site detection of food safety.

[0027] (4) Significant cost advantages and ease of operation: The preparation process of Eu-Mn-HOF material is relatively simple and the raw materials used are low in cost. Only a common fluorescence spectrophotometer (equipment cost is usually less than 50,000 yuan) is needed to complete the detection, without relying on expensive large instruments and equipment. In addition, after a simple 1-hour training, operators can master the entire detection process and key points of operation, which is convenient for widespread application in grassroots food safety supervision departments, food production enterprises and other places. Attached Figure Description

[0028] Figure 1 Electron micrograph of the Eu-Mn-HOF material prepared in Example 1;

[0029] Figure 2 The Eu-Mn-HOF material, HOF material, and simulated characterization analysis diagrams prepared in Example 1 are shown.

[0030] Figure 3 Transmittance diagrams of Eu-Mn-HOF material, HOF material, and H3TATB prepared in Example 1 at different wavenumbers;

[0031] Figure 4 The fluorescence excitation and emission spectra of the Eu-Mn-HOF material prepared in Example 1 are shown below.

[0032] Figure 5 The image shows the detection experiment after optimizing the Mn²⁺ addition amount in Example 2;

[0033] Figure 6 This is a detection experiment diagram after optimizing the Eu³⁺ addition amount in Example 2;

[0034] Figure 7 The thermal curve of Eu-Mn-HOF material under heating time in Example 2;

[0035] Figure 8 The fluorescence intensity diagrams of the Eu-Mn-HOF material in different solvents in Example 3 are shown.

[0036] Figure 9The image shows the fluorescence intensity of the Eu-Mn-HOF material in Example 3 at different detection times.

[0037] Figure 10 The image shows the fluorescence intensity of the Eu-Mn-HOF material at different concentrations in Example 3.

[0038] Figure 11 The sensitivity graph for detecting fluorescence intensity at different wavelengths for different concentrations of toxin flavone;

[0039] Figure 12 Fluorescence intensity graphs of different concentrations of toxin-flavin;

[0040] Figure 13 This is a fluorescence intensity specificity diagram of the Eu-Mn-HOF material used in Example 4 to detect different substances. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present invention in any way.

[0042] Example 1

[0043] The preparation method of the Eu-Mn-HOF material described in this embodiment includes the following steps:

[0044] (1) Synthesis of HOF: 50 mg of 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (H3TATB) was dissolved in 5 mL of dimethylformamide (DMF) solution; then, 15 mL of deionized water was added to the solution; after stirring at 2000 rpm for 12 hours at room temperature, the white solid was collected by centrifugation and washed 3 times with acetone; the collected solid was further soaked in dichloromethane (CH2Cl2) solution for 3 days; finally, about 35 mg of white solid was collected by centrifugation, dried, and HOF was prepared.

[0045] (2) Synthesis of Eu-Mn-HOF material: 10 mg MnCl2·4H2O and 10 mg Eu(NO3)3·6H2O were dissolved in 10 mL ethanol; then 50 mg HOF was dispersed in the above solution under 100 W ultrasonication for 1 minute, and heated at 50 °C for 1 hour. The material was obtained by centrifugation, washed 3 times with ethanol, and dried at 50 °C overnight to prepare Eu-Mn-HOF material.

[0046] like Figures 1 to 4The characterization analysis of the Eu-Mn-HOF material shows that: X-ray diffraction (XRD) of the prepared Eu-Mn-HOF material revealed that its characteristic diffraction peaks were basically consistent with the expected characteristic peaks of the HOF framework structure, indicating that the HOF framework structure was not significantly damaged during Eu³⁺ loading and Mn²⁺ modification; fluorescence spectroscopy analysis revealed that the Eu-Mn-HOF material exhibited strong Eu³⁺ fluorescence at 619 nm. 3⁺ The characteristic emission peaks indicate that the constructed Eu-Mn-HOF material has good fluorescence properties.

[0047] Detection principle: Synergistic mechanism of Eu³⁺ fluorescence internal filtration and Mn²⁺ specific recognition.

[0048] (1) Mn²⁺ specific recognition process: Mn²⁺, as the specific recognition center of toxoflavone, has a unique interaction mechanism with toxoflavone molecules; the outer electronic structure of Mn²⁺ enables it to form stable bonds with specific functional groups in toxoflavone molecules (such as carbonyl C=O, amino-NH-, etc.) through coordination bonds or electrostatic interactions; this highly specific binding ensures the selective recognition of toxoflavone by Eu-Mn-HOF materials, effectively reducing the interference of other components in the food matrix;

[0049] (2). Eu³⁺ Fluorescence Internal Filtering Effect Principle: Toxoflavones have strong light absorption characteristics in the wavelength range of 280-400nm; such as Figure 4 As shown, the optimal excitation wavelength of Eu³⁺ in the Eu-Mn-HOF material is 314 nm, which overlaps with the absorption spectrum of flavin. When flavin binds tightly to the Eu-Mn-HOF material through the specific recognition of Mn²⁺, it acts as an internal filter, absorbing a large amount of excitation light energy from Eu³⁺. This results in a linear decrease in the characteristic fluorescence intensity of Eu³⁺ at 619 nm as the concentration of flavin increases.

[0050] Detection procedure: Dissolve 0.8 mg of Eu-Mn-HOF material in 10 mL of aqueous solution to obtain a 0.08 mg / mL Eu-Mn-HOF solution. Take 990 μL of the Eu-Mn-HOF solution in a fluorescent cuvette, add 10 μL of the corresponding solution containing flavin, mix well, and detect using a fluorescence spectrometer with a 314 nm excitation spectrum. Read the emission peak at 619 nm. Figure 12 As shown, substituting the values ​​into the linear curve yields the content of toxic flavin in the solution.

[0051] Example 2

[0052] This embodiment optimizes the synthesis conditions of the Eu-Mn-HOF material described in Example 1:

[0053] (1) Optimization of Mn²⁺ ion addition: 5 mg, 10 mg, 15 mg, and 20 mg of MnCl₂·4H₂O and 10 mg of Eu(NO₃)₃·6H₂O were accurately weighed and dissolved in 10 mL of ethanol solvent respectively; then, 50 mg of HOF was placed in the above mixed solution, dispersed under constant ultrasonic conditions for 1 minute, and heated in an oil bath at 50 °C for 1 hour; after the reaction, the target material was obtained by centrifugation, washed and purified three times with ethanol, and then dried overnight in a vacuum drying oven at 50 °C to prepare Eu-Mn-HOF material; 10 mg of the dried sample (Eu-Mn-HOF material) was placed in a centrifuge tube, and 10 mL of ultrapure water was added to prepare a 1 mg / mL stock solution. 400 μL of the stock solution was transferred and diluted to 8 mL to obtain a working solution with a concentration of 0.05 mg / mL; fluorescence spectroscopy was used to analyze the HOF at 314 nm. At the excitation wavelength, the fluorescence intensity of four materials with different Mn²⁺ ion concentrations was measured at an emission wavelength of 619 nm. The sample with the strongest fluorescence response was selected for subsequent condition optimization experiments. Figure 5 The figure shown is a detection experiment diagram after optimizing the amount of Mn²⁺ ions added in this embodiment.

[0054] (2) Optimization experiment of Eu³⁺ ion addition: First, the optimized MnCl₂・4H₂O and 5 mg, 10 mg, 15 mg, and 20 mg of Eu(NO₃)₃・6H₂O were dissolved in 10 mL of ethanol solution respectively. Then, under constant ultrasonic conditions (ultrasonic time 1 minute), 50 mg of HOF was dispersed into the above mixed solution system and heated in a constant temperature environment of 50℃ for 1 hour. After the reaction was completed, the product was obtained by centrifugation, washed and purified three times with ethanol, and finally dried overnight at 50℃. 10 mg of the dried product was accurately weighed and placed in a centrifuge tube, and 10 mL of deionized water was added to prepare a solution with a concentration of 1 mg / mL; 400 μL of this solution was further diluted to 8 mL to obtain a test solution with a concentration of 0.05 mg / mL. Under 314 nm excitation light, the fluorescence intensity signals of four materials with different Mn²⁺ ion concentrations were measured at 619 nm. The material sample with the strongest fluorescence response was selected for subsequent optimization experiments. Figure 6 The figure shown is a detection experiment diagram after optimizing the Eu³⁺ ion addition amount in this embodiment.

[0055] (3) Optimization of heating time for Eu-Mn-HOF material: The material ratios optimized in steps 1 and 2 were dissolved in 10 mL of ethanol. Then, 50 mg of HOF was dispersed in the above solution under constant sonication for 1 minute, and heated at 50°C for 0.5, 1, 1.5, 2, and 2.5 hours, respectively. The material was obtained by centrifugation, washed three times with ethanol, and dried overnight at 50°C. After dilution to a solution of 0.05 mg / mL, fluorescence detection was performed, and the material with the strongest fluorescence effect was used for further optimization. Figure 7 The figure shown is a thermal curve of the Eu-Mn-HOF material under heating time in this embodiment.

[0056] Example 3

[0057] This embodiment further examines the Eu-Mn-HOF material prepared in Example 1. The specific testing steps are as follows:

[0058] (1) Effect of solvent on the fluorescence intensity of Eu-Mn-HOF material: Solvent optimization was performed using common fluorescent solvents such as water, ethyl acetate, acetonitrile, ethanol, and methanol. 0.1 mg of Eu-Mn-HOF was dissolved in 1 mL of each of these common fluorescent solvents (water, ethyl acetate, acetonitrile, ethanol, and methanol), placed in a fluorescent cuvette, and detected using a fluorescence spectrometer with a 314 nm excitation spectrum. The emission peak at 619 nm was read, and the solvent with the strongest fluorescence effect was selected. Figure 8 As shown, Eu-Mn-HOF exhibits the strongest fluorescence intensity in aqueous solution, making water the optimal solvent.

[0059] (2) Effect of detection time on the fluorescence intensity of Eu-Mn-HOF: Eu-Mn-HOF was dissolved in a solution, and after adding flavin, the solution was left to stand for 0 min, 2 min, 4 min, 6 min, and 8 min to obtain the optimal detection time. 0.1 mg of Eu-Mn-HOF was dissolved in 1 mL of water, and after standing for 0 min, 2 min, 4 min, 6 min, and 8 min, the solution was placed in a fluorescence cuvette and detected using a fluorescence spectrometer with an excitation spectrum of 314 nm. The emission peak at 619 nm was read, and the optimal detection time was selected. Figure 9 As shown, the fluorescence intensity did not decrease significantly after 2 minutes, so the optimal detection time is 2 minutes.

[0060] (3) Effect of Eu-Mn-HOF material concentration on the fluorescence intensity of Eu-Mn-HOF: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg Eu-Mn-HOF were dissolved in 10 mL of aqueous solution to obtain solutions with concentrations of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.10 mg / mL, respectively. 1 mL of each solution was placed in a fluorescent cuvette using a 1 mL pipette, and the emission peak at 619 nm was measured using a fluorescence spectrometer with an excitation spectrum of 314 nm. The optimal material concentration was then selected. Figure 10 As shown, the fluorescence intensity is strongest at 0.08 mg / mL, so the optimal material concentration is 0.08 mg / mL.

[0061] Example 4

[0062] This embodiment involves further specific testing of the Eu-Mn-HOF material prepared in Example 1. The specific testing steps are as follows:

[0063] Dissolve 0.8 mg of Eu-Mn-HOF material in 10 mL of aqueous solution to obtain a 0.08 mg / mL Eu-Mn-HOF solution. Take 990 μL of Eu-Mn-HOF solution in a fluorescent cuvette and add 10 μL of the corresponding solution containing flavin, 9,10-anthraquinone, juglone, and Ca. 2+ Mg 2+ Na + K + Cl - SO4 2- A solution of alanine, tyrosine, histidine, and phenylalanine was mixed thoroughly and detected using a fluorescence spectrometer with an excitation spectrum of 314 nm. The emission peak at 619 nm was then read. Figure 13 As shown, detecting only toxins led to a significant decrease in Eu-Mn-HOF fluorescence intensity, while other substances did not cause a decrease in Eu-Mn-HOF fluorescence intensity. Therefore, the detection of toxins using Eu-Mn-HOF is specific.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing Eu-Mn-HOF material, characterized in that, Includes the following steps: (1) Synthesis of HOF: 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine, dimethylformamide and deionized water were mixed in a mass-volume ratio of 45~50mg:4.5~5.5mL:14~16mL. The solid was collected by centrifugation, washed, soaked, collected by centrifugation again, and dried to prepare HOF. (2) Synthesis of Eu-Mn-HOF material: MnCl2·4H2O, Eu(NO3)3·6H2O, ethanol and HOF were prepared in a mass-volume ratio of 10mg:9~11mg:9~11mL:45~55mg. MnCl2·4H2O and Eu(NO3)3·6H2O were dissolved in ethanol and then ultrasonically dispersed in HOF. The mixture was heated, centrifuged, washed and dried to prepare Eu-Mn-HOF material.

2. The method for preparing Eu-Mn-HOF material according to claim 1, characterized in that, The stirring speed in step (1) is 1900~2100 rpm, and the stirring time is 12~24 hours.

3. The method for preparing Eu-Mn-HOF material according to claim 1, characterized in that, The washing mentioned in step (1) refers to washing with the organic solvent acetone 3 to 5 times; the soaking refers to soaking with the organic solvent dichloromethane for 3 to 5 days.

4. The method for preparing Eu-Mn-HOF material according to claim 1, characterized in that, The ultrasonic dispersion time in step (2) is 1 to 5 minutes, and the ultrasonic dispersion power is 80 to 120W.

5. The method for preparing Eu-Mn-HOF material according to claim 1, characterized in that, The heating temperature in step (2) is 40~50℃, and the heating time is 1~2 hours.

6. The method for preparing Eu-Mn-HOF material according to claim 1, characterized in that, The washing mentioned in step (2) refers to washing with ethanol, an organic solvent, 3 to 5 times.

7. An Eu-Mn-HOF material, characterized in that, It is prepared according to any one of claims 1 to 6.

8. The application of the Eu-Mn-HOF material according to claim 7 in the detection of flavin in Burkholderia gladioli.

9. The application of the Eu-Mn-HOF material according to claim 8 in the detection of flavin in Burkholderia gladioli, characterized in that, The Eu-Mn-HOF material was used to detect flavin from Burkholderia gladioli at wavelengths of 280-400 nm.

10. The application of the Eu-Mn-HOF material according to claim 8 in the detection of flavin in Burkholderia gladioli, characterized in that, The Eu-Mn-HOF material was used to detect Burkholderia gladioli flavin at a mass concentration of 0.05~0.1 mg / mL.

Images