Toxin Flavin Responsive Fluorescent Film and Its Application

Abstract

The present invention discloses a patulin-responsive fluorescent film and its application. The patulin-responsive fluorescent film includes a film matrix and nitrogen-doped carbon dots distributed in the film matrix. The film matrix is formed by sodium carboxymethyl cellulose, and the nitrogen-doped carbon dots are prepared from citric acid and urea. This fluorescent film has good fluorescence stability, excellent detection performance for patulin, such as high sensitivity, low detection limit, and wide linear range, and has good accuracy and reproducibility, providing a rapid, convenient, and visual detection means for the prevention and control of contamination by Pseudomonas cocovenenans in food.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety analysis, and particularly relates to a citreoviridin-responsive fluorescent film and its application. Background Art

[0002] Nowadays, with the improvement of people's living standards, food safety issues have attracted much attention. Food safety mainly covers problems such as contamination by foodborne pathogenic bacteria, mycotoxin contamination, residues of agricultural and veterinary drugs, and illegal addition of food additives. Among them, foodborne pathogenic bacteria have been taken seriously by countries around the world due to their wide influence and high incidence. Citreoviridin is a small molecule fatty acid exotoxin produced by various bacteria such as foodborne pathogenic bacteria Pseudomonas cocovenenans, Burkholderia, and Pseudomonas glumae. It mainly exists in foods such as fermented rice noodles, tremella, and potatoes that are easily contaminated by the above-mentioned bacteria in food safety incidents. This toxin is very stable to heat and is difficult to destroy through ordinary cooking. There is also no specific antidote, and the fatality rate is as high as 40-100%.

[0003] Currently, the main detection methods for citreoviridin include spectrophotometry, thin layer chromatography, high performance liquid chromatography, and mass spectrometry. The above methods have obvious advantages in terms of sensitivity and accuracy, but their expensive equipment and cumbersome sample processing procedures make them difficult to be widely applied. Biosensor detection methods can make up for the deficiencies of the above detection methods. However, due to the lack of corresponding aptamers and antibodies for citreoviridin, there are few studies on detecting citreoviridin based on biosensors, and there is a lack of biosensors in the prior art that can achieve rapid, convenient, and highly sensitive detection of citreoviridin. Summary of the Invention

[0004] In view of the above technical requirements, the present invention proposes a citreoviridin-responsive fluorescent film and its application, aiming to achieve rapid visual detection of citreoviridin.

[0005] To achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0006] In one aspect of the present invention, a citreoviridin-responsive fluorescent film is proposed. The citreoviridin-responsive fluorescent film includes a film matrix and nitrogen-doped carbon dots distributed in the film matrix. The film matrix is formed by sodium carboxymethyl cellulose, and the nitrogen-doped carbon dots are prepared from citric acid and urea.

[0007] In some embodiments of the present invention, the thickness of the citreoviridin-responsive fluorescent film is 1 μm to 500 μm.

[0008] In some embodiments of the present invention, the particle size of the nitrogen-doped carbon dots is 1 nm to 10 nm.

[0009] In another aspect of the present invention, there is provided a method for preparing the flavomycin-responsive fluorescent film as described above, comprising the following steps:

[0010] Mix nitrogen-doped carbon dots and sodium carboxymethylcellulose in deionized water to obtain a mixed solution;

[0011] Coat the mixed solution on a substrate and dry it at 40 °C to 80 °C for 0.5 h to 2 h to obtain the responsive film.

[0012] In some embodiments of the present invention, in the mixed solution, the mass-volume ratio of the nitrogen-doped carbon dots, the sodium carboxymethylcellulose and the deionized water is 0.1 g to 1 g: 0.1 g to 5 g: 1 mL to 20 mL.

[0013] In some embodiments of the present invention, the nitrogen-doped carbon dots are prepared by the following method:

[0014] Mix citric acid and urea in deionized water according to a preset ratio to obtain a mixed solution;

[0015] Place the mixed solution in a reaction kettle for hydrothermal reaction, and the obtained hydrothermal product is successively centrifuged, filtered, dialyzed and dried to obtain the nitrogen-doped carbon dots, wherein the reaction temperature of the hydrothermal reaction is 160 °C to 220 °C, the reaction time is 2 h to 8 h, and the dialysis time is 1 day to 3 days.

[0016] In some embodiments of the present invention, in the mixed solution, the mass-volume ratio of the citric acid, the urea and the deionized water is 0.1 g to 5 g: 0.1 g to 5 g: 10 mL to 100 mL.

[0017] In another aspect of the present invention, there is provided an application of the flavomycin-responsive fluorescent film as described above in the detection of flavomycin.

[0018] In still another aspect of the present invention, there is provided a method for visual detection of flavomycin, comprising the following steps:

[0019] Provide the flavomycin-responsive fluorescent film as described above;

[0020] Drop the sample to be tested on the flavomycin-responsive fluorescent film, and then place the flavomycin-responsive fluorescent film applied with the sample to be tested under ultraviolet light for observation.

[0021] In some embodiments of the present invention, the detection method further comprises:

[0022] Prepare a series of toxin standards solutions with different concentrations. Drop the toxin standards solutions with different concentrations onto the toxin-responsive fluorescent film respectively, and then measure the fluorescence intensity to obtain the linear regression equation of the relative fluorescence intensity and the toxin concentration.

[0023] Measure the fluorescence value of the toxin-responsive fluorescent film applied with the sample to be measured, and substitute the obtained fluorescence value into the linear regression equation to obtain the toxin content in the sample to be measured.

[0024] The beneficial effects of the present invention are as follows:

[0025] By using nitrogen-doped carbon dots prepared from citric acid and urea and sodium carboxymethylcellulose, the present invention constructs a fluorescence sensing film capable of visually detecting toxin, realizing the qualitative and quantitative detection of toxin. The fluorescence film and the visual detection method of the present invention have the following advantages:

[0026] (1) The method of the present invention combines nitrogen-doped carbon dots with sodium carboxymethylcellulose. The defects provided by nitrogen in the nitrogen-doped carbon dots endow the carbon dots with excellent fluorescence properties, and a strong hydrogen bond interaction is established between sodium carboxymethylcellulose and the nitrogen-doped carbon dots, protecting the fluorescent groups from being quenched, enabling the carbon dots to generate fluorescence even under solid-state films, thus realizing simple and easy-to-observe visual monitoring of toxin.

[0027] (2) Due to the overlap between the optimal excitation light of the fluorescence film prepared in the present invention and the ultraviolet absorption of toxin, toxin will absorb the excitation light of the material. Therefore, toxin has a good fluorescence quenching effect on the fluorescence film prepared in the present invention, and biological sensing detection of toxin is realized through fluorescence changes.

[0028] (3) The fluorescence film prepared in the present invention has good fluorescence stability. The fluorescence quenching effect shows a linear relationship with the increase of toxin concentration. The linear range is 1 - 60 μM, and the detection limit is 0.45 μM. It has excellent detection performance such as high sensitivity, low detection limit and wide linear range, and the linear relationship is significant, with good accuracy, precision, selectivity and reproducibility.

[0029] (4) The detection of toxin using the fluorescence film of the present invention does not require complex pretreatment steps such as extraction and separation. The operation is simple, convenient and low-cost, solving the problems of steps such as organic solvent extraction and time consumption required by the traditional high-performance liquid chromatography method, expanding the application of fluorescence sensing based on nitrogen-doped carbon dots in toxin detection, and also providing a new rapid, convenient and visual technology for the prevention and control of Pseudomonas cocovenenans contamination in food. Description of the Drawings

[0030] Figure 1 It is the synthesis route and detection mechanism diagram of the present invention.

[0031] Figure 2 It is the transmission electron microscopy image of the nitrogen-doped carbon dots of Example 1.

[0032] Figure 3 It is the scanning electron microscopy image of the fluorescent film in Example 1.

[0033] Figure 4 It is the Fourier transform infrared spectrum of the nitrogen-doped carbon dots and the fluorescent film of Example 1.

[0034] Figure 5 It is the X-ray photoelectron spectroscopy of the chemical composition of elements and groups in the fluorescent film of Example 1. Among them, A is the full spectrum of the fluorescent film containing C, N, and O elements, B is the C1s spectrum of the fluorescent film, C is the N1s spectrum of the fluorescent film, and D is the O1s spectrum of the fluorescent film.

[0035] Figure 6 It is the fluorescence excitation and emission spectrum of the fluorescent film of Example 1.

[0036] Figure 7 It is the standard curve graph for the detection of toxoflavin in the fluorescent film of Example 1 against the standard sample.

[0037] Figure 8 It is the photo of the fluorescent film of Example 1 for the detection of toxoflavin in the standard sample under ultraviolet light.

[0038] Figure 9 It is the fluorescence intensity graph of the fluorescent films prepared using different matrices. Detailed implementation manners

[0039] To make the present invention easier to understand, the present invention will be described in detail below in conjunction with embodiments. These embodiments are only illustrative and are not limited to the application scope of the present invention.

[0040] Unless otherwise specified, the operations and processing methods involved in the present invention belong to the conventional methods in the art. Unless otherwise specified, the instruments used in the present invention are conventional instruments in the art.

[0041] Preparation of Nitrogen-Doped Carbon Dots

[0042] The present invention uses citric acid and urea as precursors and synthesizes nitrogen-doped carbon dots by a hydrothermal method.

[0043] In a preferred embodiment, 0.1 g to 5 g of citric acid and 0.1 g to 5 g of urea are added to 10 mL to 100 mL of deionized water, ultrasonically dispersed, and stirred until the citric acid and urea are completely dissolved. Then, the obtained mixed solution is placed in a high-pressure reaction kettle for hydrothermal reaction. Among them, any heating method known in the art can be used for heating, and it is heated at 160 °C to 220 °C for 2 h to 8 h to obtain a dark brown carbon dot solution.

[0044] Then, the obtained carbon dot solution is centrifuged, and the supernatant is taken. After the supernatant passes through a microporous filter membrane, it is dialyzed in a dialysis bag for 1 to 3 days, and then the obtained dialysate is freeze-dried to obtain nitrogen-doped carbon dots. Optionally, the obtained nitrogen-doped carbon dots can be collected after drying and stored at low temperature.

[0045] In some embodiments of the present invention, for the purpose of avoiding the influence of impurities, citric acid is citric acid monohydrate. In some embodiments of the present invention, for the purpose of making the obtained nitrogen-doped carbon dots have strong fluorescence intensity and good fluorescence response, the experiments of the present invention are all carried out in a PBS buffer solution with a concentration of 0.01 to 0.1 M and a pH value between 6 and 9. That is to say, in the present invention, deionized water can be replaced by a PBS buffer solution with a concentration of 0.01 to 0.1 M and a pH value between 6 and 9.

[0046] The present invention optimizes the ratio of the synthetic raw materials citric acid and urea precursors, as well as the hydrothermal reaction conditions, so that the synthesized nitrogen-doped carbon dots have strong fluorescence emission intensity and reach the peak of fluorescence intensity.

[0047] The nitrogen-doped carbon dots prepared by the present invention are safe, non-toxic, and have a sensitive fluorescence response to toxoflavin, and have good applications in aspects such as toxoflavin detection and food spoilage detection. The nitrogen-doped carbon dots of the present invention have good morphology, small particle size and uniform distribution.

[0048] In some embodiments of the present invention, the particle size of the nitrogen-doped carbon dots is 1 nm to 10 nm. The carbon dots in this size range have stronger fluorescence. As the size of the carbon dots increases, the surface defects on them will be fewer, thus affecting their fluorescence performance and applications.

[0049] Preparation of Toxoflavin-Responsive Fluorescent Films

[0050] The present invention uses sodium carboxymethyl cellulose as a film matrix, utilizes its film-forming property, mixes it with nitrogen-doped carbon dots, and obtains a toxoflavin-responsive fluorescent film after film-forming treatment.

[0051] In a preferred embodiment, 0.1 g to 1 g of nitrogen-doped carbon dots and 0.1 g to 5 g of sodium carboxymethyl cellulose are added to 1 mL to 20 mL of deionized water, and stirred at 50 °C to 100 °C for 2 h to 8 h until the sodium carboxymethyl cellulose is completely dissolved. Then, the obtained mixed solution is coated or dropped on a substrate and dried in an oven at 40 °C to 80 °C for 0.5 h to 2 h to obtain a toxoflavin-responsive fluorescent film. Among them, film formation can also be achieved by standing and natural drying.

[0052] Among them, sodium carboxymethylcellulose not only serves as a film-forming agent, but more importantly, sodium carboxymethylcellulose can establish strong hydrogen bond interactions with nitrogen-doped carbon dots, protecting the fluorescent groups of nitrogen-doped carbon dots from being quenched, enabling nitrogen-doped carbon dots to emit fluorescence even under solid films.

[0053] Among them, due to the overlap between the optimal fluorescence excitation peak of the obtained fluorescent film and the ultraviolet-visible absorption peak of patulin, patulin can absorb the excitation light of the fluorescent film through the fluorescence inner filter effect, thereby quenching its fluorescence emission. Finally, the fluorescence quenching of the film can be observed under ultraviolet light. Therefore, patulin can be detected by the change in fluorescence signal intensity before and after, and based on this, a visual fluorescence film detection for patulin is established.

[0054] In some embodiments of the present invention, the thickness of the patulin-responsive fluorescent film is 1 μm to 500 μm. An overly thin film may be washed away and become uneven after being dropped with an aqueous solution of patulin, while an overly thick film will cause patulin to penetrate, affecting the surface test results.

[0055] According to a preferred embodiment of the present invention, in the patulin-responsive fluorescent film, nitrogen-doped carbon dots and sodium carboxymethylcellulose are combined through hydrogen bonds. When separately performing fluorescence detection on the solid powder of nitrogen-doped carbon dots or the film made of sodium carboxymethylcellulose, no fluorescence emission is observed, while the prepared patulin-responsive fluorescent film shows good fluorescence emission at 385 nm, and the emission intensity reaches more than 4000 a.u., thereby enabling highly sensitive and accurate detection of patulin. By optimizing the ratio of nitrogen-doped carbon dots to sodium carboxymethylcellulose and the film-forming conditions, the obtained fluorescent film has strong fluorescence emission, thus being able to produce a sensitive fluorescence response to patulin.

[0056] The fluorescence of the fluorescent film of the present invention is not significantly affected at different temperatures of -18 °C, 4 °C, 10 °C, 25 °C, and 35 °C, and during the period of storing in the dark at 4 °C for 7 days to 6 months, the emission intensity does not change significantly, indicating that the fluorescent film has good stability.

[0057] Detection of Toxoflavin

[0058] It has been experimentally found that the fluorescent film of the present invention can produce a highly sensitive fluorescence response based on patulin, so it can effectively detect patulin in food. In some embodiments of the present invention, the specific detection method is as follows:

[0059] (1) Preparation of standard curve: Prepare an aqueous solution of toxin B with a concentration of 0.1 - 120 μM, then add a PBS solution with a concentration of 0.01 - 0.1 M. After mixing evenly, take 1 - 20 μL and evenly drop it onto the surface of the toxin B-responsive fluorescent film, and then dry it. Then, perform fluorescence emission tests. Each of the above samples and the blank were measured 3 - 11 times. Based on the fluorescence quenching of the fluorescent film at different toxin B concentrations, a standard curve was drawn, and then a linear regression equation was obtained.

[0060] Among them, in some preferred embodiments of the present invention, the fluorescence intensity of the toxin B-responsive fluorescent film decreases with the increase of the toxin B concentration. There is a clear linear relationship between △F and Ct. The linear regression equation is ΔF (a.u.) = 40.01Ct (μM) + 9.37 (R = 0.9975). The linear range is 0.7 - 60 μM. The LOD obtained from the standard deviation of 11 blanks is 0.45 μM, and the LOQ is 1.48 μM.

[0061] (2) Recovery experiment of spiked actual samples: Take dried black fungus and soak it in deionized water. Add rice flour and fermented rice noodles to water and boil. Take the soup liquid respectively, and use the standard addition method to add toxin B solutions with different standard concentrations of 0.01 - 200 μM. Take 1 - 20 μL of the above mixed solution, add a PBS solution with a concentration of 0.01 - 0.1 M, and evenly drop it onto the toxin B-responsive fluorescent film. Each of the above samples was measured three times, and the fluorescence emission peak was recorded. The content of the actual sample was obtained by substituting it into the linear regression equation, and then compared with the standard concentration.

[0062] (3) Toxin production experiment of actual samples: Conduct an actual food sample toxin production experiment on Pseudomonas cocovenenans subsp. farinofermentans Co14 and NC6 strains. After activating Co14 and NC6, inoculate them into black fungus, rice flour, and fermented rice noodle samples respectively, and place them in a constant temperature incubator at 26 °C for 0 - 72 h. During a certain period of cultivation, take the surface toxin-producing part of the sample, crush it and put it into a centrifuge tube. Add a total of 0.1 - 10 mL and a PBS solution with a concentration of 0.01 - 0.1 M in small amounts and multiple times, centrifuge to obtain the supernatant. Take 1 - 20 μL of the supernatant and apply it to the toxin B-responsive fluorescent film. Each sample was measured three times, and the fluorescence emission peak was recorded. The toxin B content of the actual sample was obtained through the linear regression equation.

[0063] It can be understood that when performing visual qualitative detection of toxin B in a sample, place the toxin B-responsive fluorescent film applied with the sample to be tested under an ultraviolet lamp, and it can be determined whether the sample to be tested contains toxin B by observing the luminescence characteristics. If the fluorescence emission brightness decreases, it indicates that the fluorescence of the fluorescent film is quenched, that is, it indicates that the sample to be tested contains toxin B. Among them, Figure 1 shows the synthesis route and detection mechanism of the fluorescent film of the present invention.

[0064] The fluorescence thin film of the present invention does not require complex pretreatment steps such as extraction and separation for the detection of patulin, and is simple, convenient to operate, and low in cost. It expands the application of fluorescence sensing based on nitrogen-doped carbon dot materials in the detection of patulin, and also provides a new rapid, convenient, and visual technology for the prevention and control of Pseudomonas cocovenenans contamination in food.

[0065] The present invention is not limited to auricularia auricula, rice flour, and yeast dough, and is also applicable to general food matrices where Pseudomonas cocovenenans can grow, such as sweet potato flour, potato flour, tremella fuciformis, etc.

[0066] The following are specific embodiments of the present invention. It should be noted that the present invention is not limited to the following specific embodiments, and all equivalent transformations made on the basis of the technical solution of this application fall within the protection scope of the present invention.

[0067] Example 1

[0068] This example discloses the preparation of a nitrogen-doped carbon dot fluorescence thin film material, specifically as follows:

[0069] Step 1, prepare nitrogen-doped carbon dots: Dissolve 1 g of citric acid and 2 g of urea in 50 mL of deionized water, and ultrasonically disperse for 10 min. Subsequently, place the mixture in a high-pressure reaction kettle and react at 190 °C for 5 h to obtain a dark brown carbon dot solution. After centrifugation, take the supernatant, filter it through a 0.45 μm microporous filter membrane, and place the product in a dialysis bag for dialysis for 48 h. Freeze-dry the dialysis solution to obtain purified dark green carbon dot powder, and store it in a refrigerator at 4 °C in the dark for later use.

[0070] Step 2, prepare the fluorescence thin film: Add 0.1 g of the nitrogen-doped carbon dots prepared in Step 1 and 0.5 g of sodium carboxymethylcellulose to 15 mL of deionized water, and mechanically stir at 80 °C for 5 h.

[0071] Apply the obtained viscous product evenly on a quartz wafer, and dry it in an oven at 70 °C for 30 min to obtain a fluorescence thin film.

[0072] Figure 2 Shown is the transmission electron microscope image of the nitrogen-doped carbon dots in this example. It can be seen that the nitrogen-doped carbon dots are spherical particle structures with a particle size of about 2.12 nm. Figure 3 Shown is the scanning electron microscope image of the fluorescence thin film in this example. The thickness of the thin film is about 106.8 μm.

[0073] Figure 4 Is the Fourier transform infrared spectrum of the nitrogen-doped carbon dots and the fluorescence thin film. The nitrogen-doped carbon dots are at 3184 cm -1 , 3012 cm -1 , 2841 cm -1 , 2358 cm -1 , 1558 cm -1The peaks at [specific wavenumber] are the stretching vibrations of O-H, N-H, C-H, C=O, and C-N respectively. At 1396 cm -1 , 983 cm -1 , 821 cm -1 , the peaks are the stretching vibrations of C-O, C-O-C, and the stretching vibration of C-O-H respectively. The results confirm the synthesis of nitrogen-doped carbon dots with various functional groups such as carboxyl, amino, and hydroxyl groups on the surface. The infrared spectrum of the fluorescent film has three additional absorption peaks at 3668 cm -1 , 2978 cm -1 , and 1053 cm -1 compared with that of the nitrogen-doped carbon dots, which are the stretching vibrations of O - H, C-H, and C-O-C respectively, indicating that sodium carboxymethyl cellulose has been successfully embedded in the nitrogen-doped carbon dots and a large number of hydrogen bonds have been formed, confirming the successful composite of nitrogen-doped carbon dots and sodium carboxymethyl cellulose.

[0074] Figure 5 are the X-ray photoelectron spectra of the chemical composition of the elements and groups in the fluorescent film. Among them, Figure 5 , A in

[0075] Figure 6 is the full spectrum of the fluorescent film containing C, N, and O elements, B is the C1s spectrum of the fluorescent film, C is the N1s spectrum of the fluorescent film, and D is the O1s spectrum of the fluorescent film. Four peaks are generated in the full spectrum at 285.2 eV, 400.5 eV, 495.6 eV, and 531.1 eV, which are C1s, N1s, Na KLL, and O1s respectively. C1s can be decomposed into three peaks, which are related to C-C (284.8 eV), C-O-C (286.4 eV), and O-C=O (288.1 eV) respectively. The N 1s spectrum is decomposed into two peaks (C=N (399.87 eV) and C-NH2 (401.62 eV) respectively). The Na KLL at 495.6 eV is the Auger electron spectrum in the XPS spectrum, which is the Auger peak of Na and is generated by the transition of the L-shell electrons of the Na atom to the K-shell hole, corresponding to the sodium carboxymethyl cellulose in the raw materials. The O1s spectrum is decomposed into two peaks, located at 533.0 eV and 532.2 eV respectively, and is related to C-O and C=O. Based on this, it shows that the synthesis of the nitrogen-doped carbon dot fluorescent film is successful.

[0076] Example 2

[0077] The difference between this example and Example 1 is that in step 1, the mass of citric acid added is 2.3 g, and the mass of urea is 0.7 g.

[0078] The morphology and characterization diagrams of the fluorescent film prepared in this example are similar to those in Example 1 and will not be given separately here.

[0079] Example 3

[0080] The difference between this example and Example 1 is that in Step 1, the mass of citric acid added is 1.5 g and the mass of urea added is 1.5 g.

[0081] The morphology and characterization diagrams of the fluorescent film prepared in this example are similar to those in Example 1 and will not be given separately here.

[0082] Example 4

[0083] The difference between this example and Example 1 is that in Step 1, the mass of citric acid added is 5 g and the mass of urea added is 5 g.

[0084] The morphology and characterization diagrams of the fluorescent film prepared in this example are similar to those in Example 1 and will not be given separately here.

[0085] Example 5

[0086] The difference between this example and Example 1 is that in Step 1, the mass of citric acid added is 0.1 g and the mass of urea added is 0.1 g.

[0087] The morphology and characterization diagrams of the fluorescent film prepared in this example are similar to those in Example 1 and will not be given separately here.

[0088] Example 6

[0089] The difference between this example and Example 1 is that in Step 2, the mass of sodium carboxymethyl cellulose is 0.1 g.

[0090] The morphology and characterization diagrams of the nitrogen-doped carbon dot fluorescent film prepared in this example are similar to those in Example 1 and will not be given separately here.

[0091] Example 7

[0092] The difference between this example and Example 1 is that in Step 2, the mass of sodium carboxymethyl cellulose is 5 g.

[0093] The morphology and characterization diagrams of the fluorescent film prepared in this example are similar to those in Example 1 and will not be given separately here.

[0094] Example 8

[0095] The difference between this example and Example 1 is that in Step 2, the mass of sodium carboxymethyl cellulose is 0.7 g.

[0096] The morphology and characterization diagrams of the fluorescent film prepared in this example are similar to those in Example 1 and will not be given separately here.

[0097] Example 9

[0098] The difference between this example and Example 1 lies in that: in Step 2, the mass of sodium carboxymethylcellulose is 2.5 g.

[0099] The morphology and characterization diagram of the fluorescent film prepared in this example are similar to those in Example 1, and will not be given separately here.

[0100] Example 10

[0101] Using the fluorescent film prepared in Example 1 above, the visualization detection of patulin in food was carried out as follows:

[0102] (1) Plotting of the standard curve: Prepare an aqueous solution of patulin with a concentration of 0.1 - 120 μM, add 0.1 M PBS solution, gently shake, and after mixing evenly, take 7 μL and evenly drop it on the surface of the fluorescent film and dry it, then perform fluorescence testing. Each of the above samples and the blank were measured 3 - 11 times. Based on the fluorescence quenching of the film at different patulin concentrations, a standard curve was plotted. And take its fluorescence photo under ultraviolet light.

[0103] (2) Spiking recovery experiment of actual samples: Take dried black fungus and soak it in deionized water, boil rice flour and fermented rice noodles in water, take the soup liquid respectively, and use the standard addition method to add patulin solutions with different standard concentrations of 0.01 - 120 μM. Take the above mixed solution and add it to a cuvette, then add 0.1 M PBS solution and gently shake, take 7 μL and evenly drop it on the surface of the fluorescent film and dry it. Each of the above samples was measured three times, record the fluorescence emission peak, obtain the content of the actual sample by substituting into the linear curve, and then compare it with the standard concentration.

[0104] (3) Toxin production experiment of actual samples: For Burkholderia gladioli NC6 and Co14 strains of Pseudomonas cocovenenans, the toxin production experiment of actual food samples was carried out. After activating Co14 and NC6, they were respectively inoculated into black fungus, rice flour, and fermented rice noodle samples, and placed in a constant temperature incubator at 26 °C for 0 - 72 h. During the cultivation process, at 12 h, 24 h, 48 h, and 72 h, take the toxigenic part of the sample, crush it and put it into a centrifuge tube, add 1 mL of deionized water and 0.1 M PBS solution in small amounts and centrifugally take the supernatant, take 7 μL and evenly drop it on the surface of the fluorescent film and dry it. Each sample was measured three times, record the fluorescence emission peak, and obtain the content of the actual sample through the linear curve.

[0105] Figure 7 It is the standard curve diagram of the detection of patulin in the reference sample by the fluorescent film in this example. Its linear range is 1 - 60 μM, and the detection limit is 0.45 μM.

[0106] Figure 8It is a photo under ultraviolet light of the nitrogen-doped carbon dot fluorescent film in this example for detecting patulin in the reference sample. The concentrations of patulin added in the first row are 0 μM, 1 μM, 2 μM, 5 μM, and 10 μM from left to right; those in the second row are 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM from left to right.

[0107] Table 1 shows the standard addition recovery experiment of the nitrogen-doped carbon dot fluorescent film in this example for patulin in actual samples.

[0108] Table 1. Standard addition recovery experiment of the nitrogen-doped carbon dot fluorescent film in this example for sensing and detecting patulin

[0109]

[0110] Table 2 shows the fluorescence detection of patulin in actual mycotoxin-producing food samples such as rice flour, fermented rice noodles, and agaric by the nitrogen-doped carbon dot fluorescent film in this example.

[0111] Table 2. Detection of patulin by the nitrogen-doped carbon dot fluorescent film in actual mycotoxin-producing food samples in this example

[0112]

[0113] It shows that this fluorescent film can be applied to the qualitative and quantitative detection of actual samples, with high sensitivity and low detection limit. The present invention provides a rapid and convenient new technology for the prevention and control of Pseudomonas cocovenenans pollution in foods and the monitoring of product contamination.

[0114] Example 11

[0115] In this example, hydroxyethyl cellulose (HMC), chitosan (CMCS), and polyethylene glycol (PEG) were respectively used as the forming materials of the film matrix to embed nitrogen-doped carbon dots. Among them, the preparation methods of each fluorescent film are the same as those in Example 1.

[0116] From Figure 9 It can be seen that only the fluorescent film prepared by embedding nitrogen-doped carbon dots into the film matrix formed by sodium carboxymethyl cellulose will produce strong fluorescence emission, while the fluorescence intensities of other fluorescent films are relatively low. This is because strong hydrogen bond interactions are established between sodium carboxymethyl cellulose and nitrogen-doped carbon dots, protecting the fluorescent groups from being quenched.

[0117] In summary, the present invention proposes a composite film material of nitrogen-doped carbon dots and sodium carboxymethyl cellulose with a highly sensitive fluorescence response to patulin. This composite film material has the advantages of convenient preparation, inexpensive reagents, simple synthesis method, high luminescence intensity, etc., realizes the qualitative and quantitative detection of patulin, and has good accuracy, precision, selectivity, and reproducibility.

[0118] The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A Toxin B-responsive fluorescent film, characterized in that, The flavotoxin-responsive fluorescent film includes a film matrix and nitrogen-doped carbon dots distributed in the film matrix. The film matrix is formed of sodium carboxymethylcellulose, and the nitrogen-doped carbon dots are prepared from citric acid and urea. Among them, the thickness of the flavotoxin-responsive fluorescent film is 1 μm to 500 μm.

2. The flavomycin-responsive fluorescent film according to claim 1, wherein The particle size of the nitrogen-doped carbon dots is 1 nm to 10 nm.

3. A method for preparing a tioxanthin-responsive fluorescent thin film according to any one of claims 1 to 2, characterized in that, It includes the following steps: Mix nitrogen-doped carbon dots and sodium carboxymethylcellulose in deionized water to obtain a mixed solution. Coat the mixed solution on a substrate and dry it at 40 °C to 80 °C for 0.5 h to 2 h to obtain the responsive film.

4. The preparation method according to claim 3, wherein In the mixed solution, the mass-volume ratio of the nitrogen-doped carbon dots, the sodium carboxymethylcellulose, and the deionized water is 0.1 g to 1 g: 0.1 g to 5 g: 1 mL to 20 mL.

5. The preparation method according to claim 3, wherein The nitrogen-doped carbon dots are prepared by the following method: Mix citric acid and urea in deionized water according to a preset ratio to obtain a mixed solution. Place the mixed solution in a reaction kettle for hydrothermal reaction. The obtained hydrothermal product is successively centrifuged, filtered, dialyzed, and dried to obtain the nitrogen-doped carbon dots. Among them, the reaction temperature of the hydrothermal reaction is 160 °C to 220 °C, the reaction time is 2 h to 8 h, and the dialysis time is 1 day to 3 days.

6. The preparation method according to claim 5, characterized in that, In the mixed solution, the mass-volume ratio of the citric acid, the urea, and the deionized water is 0.1 g to 5 g: 0.1 g to 5 g: 10 mL to 100 mL.

7. Use of the flavotoxin-responsive fluorescent film according to any one of claims 1 to 2 in detecting flavotoxin.

8. A method for visual detection of Toxin A, characterized in that, It includes the following steps: Provide the flavotoxin-responsive fluorescent film according to any one of claims 1 to 2. Drop the sample to be tested on the flavotoxin-responsive fluorescent film, and then place the flavotoxin-responsive fluorescent film applied with the sample to be tested under ultraviolet light for observation.

9. The detection method according to claim 8, wherein It also includes: Prepare a series of flavotoxin standard solutions with different concentrations, respectively drop the flavotoxin standard solutions with different concentrations on the flavotoxin-responsive fluorescent film, then measure the fluorescence intensity, and obtain the linear regression equation of the relative fluorescence intensity and the flavotoxin concentration. Measure the fluorescence value of the flavotoxin-responsive fluorescent film applied with the sample to be tested, and substitute the obtained fluorescence value into the linear regression equation to obtain the flavotoxin content in the sample to be tested.

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