Preparation method of ratio fluorescent chromatographic test strip and application in detection of patulin

Abstract

The application discloses a preparation method of a ratio fluorescent chromatography test strip and application in detection of patulin, and comprises the following steps: S1, preparation of blue carbon dots; S2, preparation of gold nanoclusters; S3, preparation of a ratio fluorescent probe. The application is based on a strategy of red fluorescence quenching and blue fluorescence opening. The solution of thiol functionalized AuNCs is rich in functional groups (-SH) and is combined with patulin. Red fluorescence is gradually quenched, and blue fluorescence is opened, so that the function of detecting patulin is realized. The change of fluorescence color is from red to blue, and the whole fluorescence signal response can be completed within 10 seconds. In addition, with the continuous increase of the concentration of patulin, the red fluorescence is gradually quenched, the blue fluorescence is gradually enhanced, and a series of changes in fluorescence intensity is presented under an ultraviolet lamp, so that the visual detection of patulin is realized. The application can realize convenient and rapid preliminary detection of food health.

Description

Technical Field

[0001] The invention relates to a preparation method of a ratio fluorescence chromatography test strip and application thereof in patulin detection, belonging to the technical field of food detection. Background Art

[0002] Patulin is a water-soluble, heat-stable, and highly toxic mycotoxin widely found in rotting fruits, juices, and crops. Consuming food contaminated with patulin (PAT) can lead to acute and chronic toxicity and potential long-term health risks. Given the widespread presence and high toxicity of patulin in food, the development of a simple, rapid, and sensitive detection method is essential.

[0003] Immunoassays are highly efficient and universal mycotoxin detection techniques. Li et al. achieved sensitive detection of PAT using a FAM-Apt / Cr(OH)3 aptamer sensor. However, the difficulty in obtaining antigens and antibodies limits their potential for future practical applications, such as the construction of portable fluorescence sensing devices. Currently, with the rise of carbon dots (CDs), quantum dots (QDs), gold nanoclusters (AuNCs), and various fluorescent materials, fluorescence analysis has attracted considerable attention due to its excellent sensitivity, flexibility, and selectivity. Furthermore, surface chemical engineering of these fluorescent materials has also shown significant applications. Liu et al. synthesized thiol-functionalized nitrogen-doped carbon dots (CDs) by reacting CDs with L-cysteine ​​through an amide reaction, developing an "on" sensor for PAT detection. Li et al. designed a small molecule fluorescent probe, FITC-Lys, which reacts with the Lys group to form a fluorescein dimer, enabling visual fluorescence detection of PAT. While these strategies make fluorescence spectrometers sensitive and rapid for PAT detection, monochromatic fluorescence sensing, which relies on changes in the intensity of a single fluorescent light, is unreliable because intensity is easily affected by environmental factors and probe concentration. Ratiometric fluorescence sensing, which leverages the human eye's greater sensitivity to changes in color than to changes in brightness, improves detection accuracy and overcomes these limitations. Summary of the Invention

[0004] In response to the above-mentioned problems in the prior art, the present invention provides a method for preparing a ratiometric fluorescence chromatography test strip and its application in patulin detection, thereby realizing convenient and rapid preliminary detection of food health.

[0005] In order to achieve the above object, the present invention adopts a method for preparing a ratiometric fluorescent probe, comprising the following steps:

[0006] S1. Preparation of blue carbon dots

[0007] Sodium citrate and polyacrylamide were dissolved in distilled water, after being mixed uniformly, the mixture was transferred to a reaction kettle with a polytetrafluoroethylene lining, the reaction was heated, after the reaction was completed, the reaction kettle was cooled to room temperature, the solution was dialyzed for purification, and the blue carbon dot powder was dried in an oven to obtain a B-CDs solution, which was placed in a refrigerator for use;

[0008] S2, preparation of gold nanoclusters

[0009] S201, GSH was completely dissolved in water, then HAuCl4 solution was added, the mixed solution was reacted at room temperature to form GSH – Au + solution;

[0010] S202, MUA and NaOH were added to water to form a MUA solution, and the GSH – Au + solution prepared in step S201 was added to the MUA solution, the mixed solution formed emitted orange-red fluorescence under ultraviolet light, and the solution after aging at room temperature was transferred to a dialysis bag and dialyzed in ultrapure water to obtain a MUA-AuNCs solution after purification;

[0011] S3, preparation of a ratio fluorescent probe

[0012] The B-CDs solution prepared in step S1 and the MUA-AuNCs solution prepared in step S2 were taken, mixed thoroughly, and diluted with PBS buffer to obtain a fluorescent probe solution.

[0013] As an improvement, in step S1, 0.5-2 g of sodium citrate and 0.3-1 g of polyacrylamide were dissolved in 10-30 mL of distilled water; the reaction kettle was heated to 180-220°C for 2-4 h; and the concentration of the prepared B-CDs solution was 0.5-2 mg / mL.

[0014] As an improvement, in step S201, 80-100 mg of GSH was completely dissolved in 10-20 mL of water, then 4-6 mL of 1% HAuCl4 solution was added, the mixed solution was reacted at room temperature for 20-40 minutes to form GSH – Au + solution;

[0015] In step S202, 13-14 mg of MUA and 0.2-0.4 mL of 1M NaOH were added to 14-16 mL of water to form a MUA solution, and 3-5 mL of the GSH – Au + solution prepared in step S201 was added.

[0016] As an improvement, in step S3, 10 μL of the B-CDs solution prepared in step S1 and 60 μL of the MUA-AuNCs solution prepared in step S2 were taken, mixed thoroughly, and diluted to 2 mL with 10 mM, pH = 7.4 PBS buffer to obtain a fluorescent probe solution.

[0017] As an improvement, the following steps are specifically included:

[0018] S1. Preparation of blue carbon dots

[0019] Dissolve 1 g of sodium citrate and 0.52 g of polyacrylamide in 20 mL of distilled water and stir for at least 1 hour. After mixing evenly, transfer the mixture to a polytetrafluoroethylene-lined reactor and heat to 200°C for 3 hours. After the reaction is complete, cool the reactor to room temperature and purify the solution by dialyzing it in a dialysis bag for 4 hours. Dry it in a 60°C oven to obtain blue carbon dot powder. Dissolve the blue carbon dot powder in distilled water to obtain a 1 mg / mL B-CDs solution, which is then placed in a refrigerator for later use.

[0020] S2. Preparation of gold nanoclusters

[0021] S201. Dissolve 92.2 mg of GSH completely in 14.9 mL of water, ultrasonicate for at least 0.5 h, stir for at least 0.5 h, then add 5.1 mL of 1% HAuCl4 solution, and react the mixture at room temperature for 30 min to form GSH. – Au + solution;

[0022] S202, 13.1 mg MUA and 0.3 mL 1M NaOH were added to 15.7 mL water to form a MUA solution, and 4 mL of GSH prepared in step S201 was added to the MUA solution. – Au + The solution was ultrasonicated for no less than 0.5 hours and stirred for no less than 0.5 hours, and the resulting mixed solution emitted orange-red fluorescence under ultraviolet light. The solution was aged at room temperature for 5 hours, and the aged solution was transferred to a dialysis bag for dialysis, and dialyzed in ultrapure water for more than 24 hours. After purification, a MUA-AuNCs solution was obtained.

[0023] S3. Preparation of ratiometric fluorescent probe

[0024] Take 10 μL of the B-CDs solution prepared in step S1 and 60 μL of the MUA-AuNCs solution prepared in step S2, mix them thoroughly, and dilute them to 2 mL with 10 mM PBS buffer, pH = 7.4.

[0025] The second aspect of the present application provides a ratio fluorescent probe prepared by the preparation method.

[0026] The third aspect of the present application provides a ratio fluorescent chromatography test strip, wherein the fluorescent probe solution prepared by the preparation method is repeatedly sprayed on the cellulose membrane by a slide film gold spraying instrument to prepare the ratio fluorescent chromatography test strip, and the ratio fluorescent chromatography test strip is dried for standby use.

[0027] The fourth aspect of the present application provides an application of the ratio fluorescent chromatography test strip in detection of patulin, and the ratio fluorescent chromatography test strip is used for visual detection of patulin.

[0028] As an improvement, under the excitation light at 340 nm, the fluorescent spectrum is in the range of 400-800 nm, 0.5 mL of the patulin solution with a concentration of 0.1-2.2 μM is added dropwise on the ratio fluorescent chromatography test strip, the red fluorescence of the test line of the chromatography test strip is quenched, and the blue fluorescence is turned on.

[0029] As an improvement, the ratio fluorescent chromatography test strip is used for visual detection of patulin in food.

[0030] The present application is based on the strategy of red fluorescence quenching and blue fluorescence turning on, the solution of the thiol functionalized AuNCs is rich in functional groups (-SH) and is combined with patulin, the red fluorescence is gradually quenched, and the blue fluorescence is turned on, so that the function of detecting patulin is realized, the change of the fluorescence color is from red to blue, and the whole fluorescence signal response can be completed within 10 seconds; in addition, with the increasing concentration of patulin, the red fluorescence is gradually quenched, and the blue fluorescence is gradually enhanced, and a series of changes in the fluorescence intensity is presented under the ultraviolet lamp, so that the visual detection of patulin is realized.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] 1. The ratio fluorescent probe and the ratio fluorescent chromatography test strip are prepared by combining the thiol functionalized gold clusters (MUA-AuNCs) with the blue carbon dots (B-CDs), a red fluorescence quenching and blue fluorescence turning on type system is formed, the system has good selectivity and sensitivity to patulin, can effectively avoid the interference of other impurities, and has a rapid response; the ratio fluorescent probe is used for detecting patulin, the dual-color fluorescence intensity is relatively stable, and the visual detection is realized.

[0033] 2. The response time of the ratio fluorescent probe and the ratio fluorescent chromatography test strip is completed within 10 seconds, rapid detection is realized, and the detection limit of patulin is 0.019 μM.

[0034] 3. The ratio fluorescent chromatography test strip is safe and stable, and is convenient to carry, and the time and space limitations are broken. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 (A) is a transmission electron microscopy image of the fluorescent probe solution of Example 1 of the present invention;

[0036] Figure 2 (A) is a transmission electron micrograph of the fluorescent probe solution of Example 1 of the present invention after adding patulin;

[0037] Figure 3 The fluorescence spectra and color change diagrams of the ratio fluorescence chromatography test strips with different concentrations of patulin. With the increase of patulin concentration, the fluorescence color of PAT (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 μM) solution gradually changes from red to blue.

[0038] Figure 4 Schematic diagram of the ratiometric fluorescence chromatography test strip for detecting patulin in actual samples;

[0039] Figure 5 For I 450 / I 620 Calibration curve between PAT (0-2.2 μM) concentrations;

[0040] Figure 6 To study the selectivity of the ratiometric fluorescent probe, metal ions (K + Mg 2+ 、Fe 2+ ), anion (Br - 、F - ), vitamin B (VB), vitamin C (VC), lysine (Lys), and mycotoxins (FB1), (DON), (OTA), and (AFB1) fluorescence responses of the ratiometric fluorescence probe. DETAILED DESCRIPTION

[0041] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0042] Example 1

[0043] A method for preparing a ratiometric fluorescent probe comprises the following steps:

[0044] S1. Preparation of blue carbon dots

[0045] Dissolve 1 g of sodium citrate and 0.52 g of polyacrylamide in 20 mL of distilled water and stir for at least 1 hour. After mixing evenly, transfer the mixture to a 50 mL polytetrafluoroethylene-lined reactor and heat to 200°C for 3 hours. After the reaction is complete, cool the reactor to room temperature and dialyze the solution in a dialysis bag (1000 Da) for 4 hours for purification. Dry the solution in a 60°C oven to obtain blue carbon dot powder. Dissolve the blue carbon dot powder in distilled water to obtain a 1 mg / mL B-CDs solution, which is then stored in a 4°C refrigerator for later use.

[0046] S2. Preparation of gold nanoclusters

[0047] S201. Dissolve 92.2 mg of GSH (glutathione) completely in 14.9 mL of water, ultrasonicate for at least 0.5 h, stir for at least 0.5 h, then add 5.1 mL of 1% HAuCl4 solution, and react the mixture at room temperature for 30 min to form GSH. – Au + During this process, the light brown solution gradually turns to light yellow and finally becomes almost colorless;

[0048] S202, 13.1 mg MUA (11-mercaptoundecanoic acid) and 0.3 mL 1M NaOH were added to 15.7 mL water to form a MUA solution, and 4 mL of GSH prepared in step S201 was added to the MUA solution. – Au + The solution was ultrasonicated for no less than 0.5 h and stirred for no less than 0.5 h. The resulting mixed solution emitted orange-red fluorescence under UV light within a few seconds and was aged at room temperature for 5 h without stirring. During this period, the solution remained colorless. The aged solution was transferred to a dialysis bag (molecular weight cutoff of 8-14 kDa) and dialyzed in ultrapure water for more than 24 h. The purified MUA-AuNCs solution could be stored at 4 °C for 3 months with negligible changes in its optical properties.

[0049] S3. Preparation of ratiometric fluorescent probe

[0050] Take 10 μL of the B-CDs solution prepared in step S1 and 60 μL of the MUA-AuNCs solution prepared in step S2, mix them thoroughly, and dilute them to 2 mL with 10 mM, pH = 7.4 PBS buffer to obtain the fluorescent probe solution.

[0051] The transmission electron microscope image of the fluorescent probe solution of Example 1 is as follows: Figure 1 As shown in Figure 2, MUA-AuNCs have uniform morphology and good dispersion, with a particle size of about 2 nm; the transmission electron microscopy image of patulin added, as shown in Figure 2Figure 2 As shown in the figure, analysis shows that after adding a certain amount of PAT (patulin), the action of the surface groups of MUA-AuNCs causes particle aggregation, and PAT acts on the surface of AuNCs through covalent bonds, increasing the particle size to about 2-4 nm.

[0052] The preparation steps of the ratio fluorescence chromatography test strip are as follows: take the above-mentioned fluorescent probe solution and place it in a gold spray film scratcher. The gold spray film scratcher is driven by a stepping pump, and the liquid output accuracy is controlled at 0.01 uL. The stepping pump discharges the fluorescent probe solution while spraying it, and the fluorescent probe solution is sprayed out of the nozzle. Quantitative spray points are marked on the fiber membrane at intervals of 6 mm. The process is repeated 50 times in a linear manner to achieve a significant effect. The ratio fluorescence chromatography test strip is then dried under ambient conditions for 24 hours and set aside.

[0053] Patulin solution test:

[0054] (1) Detection of patulin solution by fluorescent probe solution

[0055] 100 μM metal ions (K + Mg 2+ 、Fe 2+ ), anion (Br - 、F - ), vitamin B (VB), vitamin C (VC), lysine (Lys), and 50 μM of the fungal toxins (FB1), (DON), (OTA), and (AFB1) were used to study the fluorescence response of the ratiometric fluorescent probes for patulin and patulin mixtures, as well as some ions and lysine. Fluorescence spectra of the probe solutions were recorded in the 400-800 nm range using 340 nm excitation. The addition of patulin revealed a sharp quenching of the red color and a dramatic enhancement of the blue color.

[0056] To the fluorescent probe solution, 0.5 mL of patulin solution at different concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, and 2.2 μM) was added. The probe solution was placed in separate vials and the corresponding fluorescence spectra were recorded at 340 nm as the patulin concentration increased. By establishing a relationship between fluorescence intensity and patulin concentration, the detection limit for patulin was determined to be 0.019 μM.

[0057] (2) Detection of patulin solution using ratiometric fluorescence chromatography test strips

[0058] 0.5 mL of patulin solution of different concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 μM) was added to the fluorescent probe solution, and the solutions were placed in different vials. At 340 nm excitation light, the corresponding fluorescence spectra were recorded by orderly excitation as the patulin concentration increased.

[0059] The prepared ratiometric fluorescence chromatographic test strips were used for practical application in the determination of patulin. Patulin solutions of varying concentrations (0-50 μM) were sequentially dripped onto different ratiometric fluorescence chromatographic test strips. As the patulin concentration increased, the color intensity decreased, and the ratiometric fluorescence chromatographic test strips showed a clear change from red to blue.

[0060] To the fluorescent probe solution prepared in Example 1, different concentrations of patulin solution were added, mixed, and the fluorescence intensity was tested. The results showed that the blue fluorescence emission peak at 450 nm gradually became stronger, and the red fluorescence emission peak at 620 nm gradually became weaker. 450 / I 620 The calibration graph shows that the fluorescence intensity ratio is proportional to the concentration of patulin, and the linear regression curve of y = -0.00838x + 1.0468 is fitted, which provides the correlation, such as Figure 5 As shown in Figure 2, quantitative detection of patulin solution can be achieved. When the excitation light is 340 nm, the fluorescence spectrum of the mixed system in the wavelength range of 400-800 nm is recorded, as shown in Figure 2. Figure 3 shown.

[0061] Patulin solutions of different concentrations were added to different actual samples, and then the sample solutions were added to the ratio fluorescence chromatography test strips. The reaction lasted for 10 minutes, and the ratio fluorescence chromatography test strips were placed on the detection platform. Fluorescence images were obtained under a 365nm UV lamp in a dark environment. The specific concentration of patulin was read, such as Figure 4 shown.

[0062] In order to explore the interference of the probe system, the selectivity study was carried out by analyzing the fluorescence properties of these probe systems to determine patulin. 100 μM metal ions (K + Mg 2+ 、Fe 2+ ), anion (Br - 、F - ), vitamin B (VB), vitamin C (VC), lysine (Lys), and 50 μM fungal toxins (FB1), (DON), (OTA), and (AFB1) were used to study the fluorescence response of patulin and patulin mixtures as well as partial ions and lysine ratio fluorescence probes, such as Figure 6The fluorescence spectrum of the probe solution was recorded in the 400-800 nm range using 340 nm excitation light. The addition of patulin revealed a sharp quenching of the red fluorescence and a sharp enhancement of the blue fluorescence. Introducing the complementary interfering species into the probe system did not show any spectral changes. Adding patulin quenched the red fluorescence, enhanced the blue fluorescence, and significantly changed the intensity ratio. These results demonstrate the system's excellent selectivity and anti-interference properties for patulin.

[0063] Example 2

[0064] A method for preparing a ratiometric fluorescent probe comprises the following steps:

[0065] S1. Preparation of blue carbon dots

[0066] Dissolve 0.5 g of sodium citrate and 0.3 g of polyacrylamide in 10 mL of distilled water. After mixing, transfer the mixture to a polytetrafluoroethylene-lined reactor and heat to 180°C for 2 h. After the reaction is completed, cool the reactor to room temperature, dialyze the solution for purification, and dry it in an oven to obtain blue carbon dot powder. Dissolve the blue carbon dot powder in distilled water to obtain a B-CDs solution with a concentration of 0.5 mg / mL, which is then placed in a refrigerator for later use.

[0067] S2. Preparation of gold nanoclusters

[0068] S201. 80 mg of GSH was completely dissolved in 10 mL of water, and then 4 mL of 1% HAuCl4 solution was added. The mixed solution was reacted at room temperature for 20 minutes to form GSH. – Au + solution;

[0069] S202, 13 mg MUA and 0.2 mL 1M NaOH were added to 14 mL water to form a MUA solution, and 3 mL of GSH prepared in step S201 was added to the MUA solution. – Au + The resulting mixed solution emitted orange-red fluorescence under ultraviolet light and was aged at room temperature for 4 hours. The aged solution was transferred to a dialysis bag and dialyzed in ultrapure water to obtain a MUA-AuNCs solution after purification.

[0070] S3. Preparation of ratiometric fluorescent probe

[0071] Take 10 μL of the B-CDs solution prepared in step S1 and 60 μL of the MUA-AuNCs solution prepared in step S2, mix them thoroughly, and dilute them to 2 mL with 10 mM, pH = 7.4 PBS buffer to obtain the fluorescent probe solution.

[0072] The preparation steps of the ratio fluorescence chromatography test strip are as follows: take the above-mentioned fluorescent probe solution and place it in a gold spray film scratcher. The gold spray film scratcher is driven by a stepping pump, and the liquid output accuracy is controlled at 0.01 uL. The stepping pump discharges the fluorescent probe solution while spraying it, and the fluorescent probe solution is sprayed out of the nozzle. Quantitative spray points are marked on the fiber membrane at intervals of 6 mm. The process is repeated 50 times in a linear manner to achieve a significant effect. The ratio fluorescence chromatography test strip is then dried under ambient conditions for 24 hours and set aside.

[0073] Example 3

[0074] A method for preparing a ratiometric fluorescent probe comprises the following steps:

[0075] S1. Preparation of blue carbon dots

[0076] Dissolve 2 g of sodium citrate and 1 g of polyacrylamide in 30 mL of distilled water. After mixing, transfer the mixture to a polytetrafluoroethylene-lined reactor and heat to 220°C for 4 h. After the reaction is completed, cool the reactor to room temperature, dialyze the solution for purification, and dry it in an oven to obtain blue carbon dot powder. Dissolve the blue carbon dot powder in distilled water to obtain a B-CDs solution with a concentration of 2 mg / mL, which is then placed in a refrigerator for later use.

[0077] S2. Preparation of gold nanoclusters

[0078] S201. Dissolve 100 mg of GSH completely in 20 mL of water, then add 6 mL of 1% HAuCl4 solution, and react the mixed solution at room temperature for 40 minutes to form GSH. – Au + solution;

[0079] S202, 14 mg MUA and 0.4 mL 1M NaOH were added to 16 mL water to form a MUA solution, and 5 mL of GSH prepared in step S201 was added to the MUA solution. – Au + The resulting mixed solution emitted orange-red fluorescence under ultraviolet light and was aged at room temperature for 6 hours. The aged solution was transferred to a dialysis bag and dialyzed in ultrapure water to obtain a MUA-AuNCs solution after purification.

[0080] S3. Preparation of ratiometric fluorescent probe

[0081] Take 10 μL of the B-CDs solution prepared in step S1 and 60 μL of the MUA-AuNCs solution prepared in step S2, mix them thoroughly, and dilute them to 2 mL with 10 mM, pH = 7.4 PBS buffer to obtain the fluorescent probe solution.

[0082] The preparation steps of the ratio fluorescent chromatography test strip are as follows: the fluorescent probe solution is placed in a gold spraying and film drawing instrument, the instrument is driven by a stepping pump, the liquid output precision is controlled to be 0.01 uL, the fluorescent probe solution is discharged by the stepping pump when the fluorescent probe solution is sprayed, the fluorescent probe solution is sprayed from the spraying port, the quantitative spraying points are marked at intervals of 6 mm on the fiber membrane, the process is repeated 50 times to achieve obvious effect, and then the ratio fluorescent chromatography test strip is dried for 24 hours under ambient conditions and is ready for use.

[0083] Example 4

[0084] In different actual samples (apple juice, tomato juice, peanut juice and wheat juice), different concentrations of patulin solution are added dropwise, then the sample solution is added dropwise to the ratio fluorescent chromatography test strip, reaction is performed for 10 min, the ratio fluorescent chromatography test strip is placed into a detection platform, and a fluorescence photo is acquired by a 365 nm ultraviolet lamp in a dark environment. The specific concentration value of patulin is read.

[0085] The above only describes the preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a ratiometric fluorescent probe, characterized in that: The following steps are involved: S1. Preparation of blue carbon dots Sodium citrate and polyacrylamide were dissolved in distilled water and mixed well. The mixture was transferred to a polytetrafluoroethylene-lined reactor and heated for reaction. After the reaction was completed, the reactor was cooled to room temperature, the solution was dialyzed for purification, and dried in an oven to obtain blue carbon dot powder. The blue carbon dot powder was dissolved in distilled water to obtain a B-CDs solution, which was placed in a refrigerator for use. S2. Preparation of gold nanoclusters S201, GSH is completely dissolved in water, and then HAuCl4 solution is added, and the mixed solution is reacted at room temperature to form GSH – Au + solution; S202, MUA and NaOH are added to water to form a MUA solution, and the GSH prepared in step S201 is added to the MUA solution. – Au + The resulting mixed solution emits orange-red fluorescence under ultraviolet light and is aged at room temperature. The aged solution is transferred to a dialysis bag and dialyzed in ultrapure water to obtain a MUA-AuNCs solution after purification. S3. Preparation of ratiometric fluorescent probe The B-CDs solution prepared in step S1 and the MUA-AuNCs solution prepared in step S2 were mixed thoroughly and diluted with PBS buffer to obtain a fluorescent probe solution.

2. The method for preparing a ratiometric fluorescent probe according to claim 1, wherein: In step S1, 0.5-2 g of sodium citrate and 0.3-1 g of polyacrylamide are dissolved in 10-30 mL of distilled water; the reactor is heated to 180-220° C. and kept warm for 2-4 hours; and the concentration of the prepared B-CDs solution is 0.5-2 mg / mL.

3. The method for preparing a ratiometric fluorescent probe according to claim 1, wherein: In step S201, 80-100 mg of GSH is completely dissolved in 10-20 mL of water, and then 4-6 mL of 1% HAuCl4 solution is added. The mixed solution is reacted at room temperature for 20-40 minutes to form GSH. – Au + solution; In step S202, 13-14 mg of MUA and 0.2-0.4 mL of 1M NaOH were added to 14-16 mL of water to form a MUA solution, and 3-5 mL of GSH prepared in step S201 was added to the MUA solution. – Au + solution.

4. The method for preparing a ratiometric fluorescent probe according to claim 1, wherein: In step S3, 10 μL of the B-CDs solution prepared in step S1 and 60 μL of the MUA-AuNCs solution prepared in step S2 were taken, mixed thoroughly, and diluted to 2 mL with 10 mM PBS buffer (pH = 7.4) to obtain a fluorescent probe solution.

5. The method for preparing a ratiometric fluorescent probe according to claim 1, wherein: The specific steps include: S1. Preparation of blue carbon dots Dissolve 1 g of sodium citrate and 0.52 g of polyacrylamide in 20 mL of distilled water and stir for at least 1 hour. After mixing evenly, transfer the mixture to a polytetrafluoroethylene-lined reactor and heat to 200°C for 3 hours. After the reaction is complete, cool the reactor to room temperature and purify the solution by dialyzing it in a dialysis bag for 4 hours. Dry it in a 60°C oven to obtain blue carbon dot powder. Dissolve the blue carbon dot powder in distilled water to obtain a 1 mg / mL B-CDs solution, which is then placed in a refrigerator for later use. S2. Preparation of gold nanoclusters S201. Dissolve 92.2 mg of GSH completely in 14.9 mL of water, ultrasonicate for at least 0.5 h, stir for at least 0.5 h, then add 5.1 mL of 1% HAuCl4 solution, and react the mixture at room temperature for 30 min to form GSH. – Au + solution; S202, 13.1 mg MUA and 0.3 mL 1M NaOH were added to 15.7 mL water to form a MUA solution, and 4 mL of GSH prepared in step S201 was added to the MUA solution. – Au + The solution was ultrasonicated for no less than 0.5 hours and stirred for no less than 0.5 hours, and the resulting mixed solution emitted orange-red fluorescence under ultraviolet light. The solution was aged at room temperature for 5 hours, and the aged solution was transferred to a dialysis bag for dialysis, and dialyzed in ultrapure water for more than 24 hours. After purification, a MUA-AuNCs solution was obtained. S3. Preparation of ratiometric fluorescent probe Take 10 μL of the B-CDs solution prepared in step S1 and 60 μL of the MUA-AuNCs solution prepared in step S2, mix them thoroughly, and dilute them to 2 mL with 10 mM PBS buffer, pH = 7.

4.

6. A ratiometric fluorescent probe, characterized in that The method is prepared according to any one of claims 1 to 5.

7. A ratiometric fluorescence chromatography test strip, characterized in that: The fluorescent probe solution prepared by the preparation method according to any one of claims 1 to 5 is repeatedly sprayed on a cellulose membrane by a synovial gold spraying apparatus to prepare a ratio fluorescence chromatography test strip, which is then dried for later use.

8. Use of the ratiometric fluorescence chromatography test strip according to claim 7 in the detection of patulin, characterized in that: Used for visual detection of patulin.

9. Use of a ratiometric fluorescence chromatography test strip in the detection of patulin according to claim 8, characterized in that: Under 340 nm excitation light, the fluorescence spectrum is in the range of 400-800 nm. When 0.5 mL of 0.1-2.2 μM patulin solution is added to the ratiometric fluorescence chromatography test strip, the red fluorescence of the test line of the chromatography test strip is quenched and the blue fluorescence is turned on.

10. Use of a ratiometric fluorescence chromatography test strip in the detection of patulin according to claim 8, characterized in that: Used for visual detection of patulin in food.

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