Preparation method of ratio fluorescence chromatography test strip and application of ratio fluorescence chromatography test strip in patulin detection

Abstract

The invention discloses a preparation method of a ratio fluorescence chromatography test strip and application of the ratio fluorescence chromatography test strip in patulin detection. The preparation method comprises the following steps: S1, preparing blue carbon dots; s2, preparing a gold nanocluster; and S3, preparing the ratiometric fluorescent probe. On the basis of a strategy of red fluorescence quenching and blue fluorescence opening, a sulfydryl functionalized AuNCs solution has abundant functional groups (-SH) which are complexed with patulin, the red fluorescence is gradually quenched, and the blue fluorescence is opened, so that the function of detecting patulin is realized, and the change of the fluorescence color from red to blue is realized. The whole fluorescence signal response can be completed within 10 seconds; besides, along with continuous increase of the patulin concentration, red fluorescence is gradually quenched, blue fluorescence is gradually enhanced, and a series of fluorescence intensity changes are shown under an ultraviolet lamp, so that visual detection of patulin is realized. According to the invention, convenient and rapid preliminary food health detection can be realized.

Description

Technical Field

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

[0002] Patulin is a water-soluble, heat-stable, highly toxic fungal toxin that is widely found in rotten fruits, juices, and crops. Eating food contaminated with patulin (PAT) can lead to acute and chronic toxicity problems and long-term potential health risks. Given the widespread presence and high toxicity of patulin in food, it is necessary to develop a simple, rapid, and sensitive detection method.

[0003] Immunoassay is an efficient and universal mycotoxin detection technology. Li et al. completed sensitive detection of PAT based on FAM-Apt / Cr(OH)3 aptamer sensor. However, due to the difficulty in obtaining antigens and antibodies, there are still certain limitations in its future practical applications, such as the construction of portable fluorescent sensing devices. At present, with the rise of carbon dots (CDs), quantum dots (QDs), gold nanoclusters (AuNCs), and various fluorescent materials, fluorescence analysis has attracted much attention due to its excellent sensitivity, flexibility and selectivity. In addition, the surface chemical engineering of various fluorescent materials has also shown important applications. Liu et al. synthesized thiol-functionalized nitrogen-doped carbon dots through the amide reaction of carbon dots and L-cysteine, and developed an "on" sensor for detecting PAT. Li et al. designed a small molecule fluorescent probe FITC-Lys, which synthesized fluorescein dimers through the reaction between Lys groups and PAT, realizing the visualized fluorescence detection of PAT. Although these strategies make the fluorescence spectrometer sensitive and fast for PAT detection, the monochrome fluorescence sensing method, that is, changing the intensity of a single fluorescence, is unreliable because the intensity is easily affected by environmental and probe concentration conditions. Ratiometric fluorescence sensing improves detection accuracy by taking advantage of the fact that the human eye is much more sensitive to color changes than to brightness changes, thereby eliminating the above limitations. Summary of the invention

[0004] In view of the problems existing in the above-mentioned prior art, the present invention provides a method for preparing a ratio fluorescence chromatography test strip and its application in patulin detection, so as to realize 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, and after being mixed evenly, the mixture was transferred to a reactor lined with polytetrafluoroethylene, 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 standby use;

[0008] S2. Preparation of gold nanoclusters

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

[0010] S202, adding MUA and NaOH to water to form a MUA solution, and adding the GSH prepared in step S201 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.

[0011] S3. Preparation of ratiometric fluorescent probe

[0012] 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.

[0013] As an improvement, in the 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 h; the concentration of the obtained B-CDs solution is 0.5-2 mg / mL.

[0014] As an improvement, 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, and the mixed solution is reacted at room temperature for 20-40 minutes to form GSH. – Au + Solution;

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

[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 are 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, stir for no less than 1 hour, and after mixing evenly, transfer the mixture to a reactor lined with polytetrafluoroethylene, heat to 200°C and keep warm for 3 hours. After the reaction is completed, wait for the reactor to cool to room temperature, dialyze the solution in a dialysis bag for 4 hours for purification, and dry it in an oven at 60°C to obtain a blue carbon dot powder. Dissolve the blue carbon dot powder in distilled water to obtain a B-CDs solution with a concentration of 1 mg / mL, and place it in a refrigerator for use;

[0020] S2. Preparation of gold nanoclusters

[0021] S201, 92.2 mg GSH was completely dissolved in 14.9 mL water, the ultrasonic time was not less than 0.5 hour, the stirring time was not less than 0.5 hour, and then 5.1 mL of 1% HAuCl4 solution was added, and the mixed solution was reacted at room temperature for 30 minutes 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 subjected to ultrasonic treatment for no less than 0.5 hours and stirring for no less than 0.5 hours, the resulting mixed solution emitted orange-red fluorescence under ultraviolet light, and aged at room temperature for 5 hours. The aged solution was transferred to a dialysis bag for dialyzing, and dialyzed in ultrapure water for more than 24 hours, and the MUA-AuNCs solution was obtained after purification;

[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, pH=7.4 PBS buffer.

[0025] The second aspect of the present invention provides a ratiometric fluorescent probe, which is prepared by the preparation method.

[0026] The third aspect of the present invention provides a ratio fluorescence chromatography test strip, wherein the fluorescent probe solution prepared by the preparation method is repeatedly sprayed on a cellulose membrane by a synovial gold spraying apparatus to prepare a ratio fluorescence chromatography test strip, and then dried for later use.

[0027] The fourth aspect of the present invention provides a use of the ratiometric fluorescence chromatography test strip in the detection of patulin, for visual detection of patulin.

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

[0029] As an improvement, it is used for visual detection of patulin in food.

[0030] The present invention is based on the strategy of quenching red fluorescence and turning on blue fluorescence. The solution of thiol-functionalized AuNCs has rich functional groups (-SH) that complex with patulin. The red fluorescence is gradually quenched and the blue fluorescence is turned on, thereby realizing the function of detecting patulin. The fluorescence color changes from red to blue, and the entire fluorescence signal response can be completed within 10 seconds. In addition, as the concentration of patulin continues to increase, the red fluorescence is gradually quenched and the blue fluorescence is gradually enhanced, presenting a series of changes in fluorescence intensity under ultraviolet light, thereby realizing the visual detection of patulin.

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

[0032] 1. Ratiometric fluorescent probes and ratiometric fluorescent chromatography test strips were prepared by combining thiol-functionalized gold clusters (MUA-AuNCs) with blue carbon dots (B-CDs), forming a red fluorescence quenching and blue fluorescence opening system. This system has good selectivity and sensitivity for patulin, can effectively avoid interference from other impurities, and has a fast response. The ratiometric fluorescence is used to detect patulin, and its dual-color fluorescence intensity is relatively stable, realizing visual detection.

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

[0034] 3. Ratio fluorescence chromatography test strips are safe, stable, easy to carry, and break the limitations of time and space. BRIEF DESCRIPTION OF THE 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 microscopy image of the fluorescent probe solution of Example 1 of the present invention with patulin added;

[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 It is a schematic diagram of detecting patulin in actual samples by using ratiometric fluorescence chromatography test strips;

[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 ratiometric fluorescent probes, metal ions (K + Mg 2+ , Fe 2+ ), anion (Br - 、F - ), vitamin B (VB), vitamin C (VC), lysine (Lys), and fungal toxins (FB1), (DON), (OTA), and (AFB1) fluorescence responses of the ratio fluorescence probe. DETAILED DESCRIPTION

[0041] The following embodiments are further descriptions of the content of the present invention as an explanation of 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, stir for no less than 1 hour, and after mixing evenly, transfer the mixture to a 50 mL reactor lined with polytetrafluoroethylene, heat to 200°C and keep warm for 3 hours. After the reaction is completed, wait for the reactor to cool to room temperature, dialyze the solution in a dialysis bag (1000Da) for 4 hours for purification, and dry it in a 60°C oven to obtain a blue carbon dot powder. Dissolve the blue carbon dot powder in distilled water to obtain a B-CDs solution with a concentration of 1 mg / mL, and place it in a 4°C refrigerator for 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 no less than 0.5 h, stir for no less than 0.5 h, then add 5.1 mL of 1% HAuCl4 solution, and react the mixed solution at room temperature for 30 min to form GSH. – Au + solution, during which 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 subjected to ultrasonication for no less than 0.5 h and stirring for no less than 0.5 h. The mixed solution formed emitted orange-red fluorescence under UV light within a few seconds. The solution was aged at room temperature for 5 h without stirring, during which time 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 was 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 a fluorescent probe solution.

[0051] The transmission electron microscopy 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 2 Figure 2 As shown, 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, 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 injection port. 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, and then the ratio fluorescence chromatography test strip is dried under ambient conditions for 24 hours for use.

[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 50μM fungal toxins (FB1), (DON), (OTA), (AFB1) were added to study the fluorescence response of patulin and patulin mixtures as well as partial ions and lysine ratio fluorescence probes. The fluorescence spectrum of the probe solution in the range of 400-800nm ​​was recorded with 340nm excitation light. When patulin was added, the red color was sharply quenched and the blue color was sharply enhanced.

[0056] 0.5 mL of patulin solutions 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) were added to the fluorescent probe solution, and placed in different vials. At 340 nm excitation light, the corresponding fluorescence spectra were recorded by sequential excitation as the concentration of patulin increased. By establishing the relationship between fluorescence intensity and patulin concentration, the detection limit of patulin was determined to be 0.019 μM.

[0057] (2) Detection of patulin solution by 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 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 ratio fluorescence chromatography test strips were used for practical application of patulin determination. Patulin solutions of different concentrations (0-50 μM) were dripped into different ratio fluorescence chromatography test strips in sequence. As the concentration of patulin increased and the color intensity decreased, the ratio fluorescence chromatography test strips showed an obvious change from red to blue.

[0060] To the fluorescent probe solution prepared in Example 1, different concentrations of patulin solution were added respectively. After mixing, 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, providing a correlation, such as Figure 5 As shown, 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 FIG. 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 test strips were reacted for 10 minutes, and the ratio fluorescence chromatography test strips were placed on the detection platform. Fluorescence photos were obtained under a 365nm ultraviolet lamp in a dark environment. The specific concentration value 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), (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 6As shown. The fluorescence spectrum of the probe solution in the range of 400-800nm ​​was recorded with 340nm excitation light. When patulin was added, the red color was quenched sharply and the blue color was enhanced sharply. The introduction of the above complementary interference species into the probe system did not show any spectral changes. When patulin was added, the red fluorescence was quenched, the blue fluorescence was turned on, and the intensity ratio changed significantly. The results showed that the system had good 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 sodium citrate and 0.3 g polyacrylamide in 10 mL distilled water, mix well, transfer the mixture to a reactor lined with polytetrafluoroethylene, heat to 180 °C for 2 h, and after the reaction is completed, wait for the reactor to cool 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, and place it in a refrigerator for 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 emits orange-red fluorescence under ultraviolet light, and is aged at room temperature for 4 hours. The aged solution is 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 a fluorescent probe solution.

[0072] The preparation steps of the ratio fluorescence chromatography test strip are as follows: take the above-mentioned fluorescent probe solution, 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 injection port. 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, and then the ratio fluorescence chromatography test strip is dried under ambient conditions for 24 hours for use.

[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, mix well, transfer the mixture to a reactor lined with polytetrafluoroethylene, heat to 220 °C for 4 h, and after the reaction is completed, wait for the reactor to cool 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, and place it in a refrigerator for 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 emits orange-red fluorescence under ultraviolet light, and is aged at room temperature for 6 hours. The aged solution is 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 a fluorescent probe solution.

[0082] The preparation steps of the ratio fluorescence chromatography test strip are as follows: take the above-mentioned fluorescent probe solution, 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 injection port. 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, and then the ratio fluorescence chromatography test strip is dried under ambient conditions for 24 hours for use.

[0083] Example 4

[0084] Patulin solutions of different concentrations were added to different actual samples (apple juice, tomato juice, peanut juice, wheat juice), and then the sample solutions were added to the ratio fluorescence chromatography test strips. After reacting for 10 minutes, the ratio fluorescence chromatography test strips were placed on the detection platform, and fluorescence photos were obtained under a 365nm ultraviolet lamp in a dark environment. The specific concentration value of patulin was read.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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 after being mixed evenly, the mixture was transferred to a reactor lined with polytetrafluoroethylene, 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 standby use; S2. Preparation of gold nanoclusters S201, GSH was completely dissolved in water, and then HAuCl4 solution was added, and the mixed solution was reacted at room temperature to form GSH – Au + Solution; S202, adding MUA and NaOH to water to form a MUA solution, and adding the GSH prepared in step S201 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, characterized in that: In the 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 h; the concentration of the obtained B-CDs solution is 0.5-2 mg / mL.

3. The method for preparing a ratiometric fluorescent probe according to claim 1, characterized in that: 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, and the mixed solution is reacted at room temperature for 20-40 minutes to form GSH. – Au + Solution; In step S202, 13-14 mg MUA and 0.2-0.4 mL 1M NaOH are added to 14-16 mL water to form a MUA solution, and 3-5 mL of GSH prepared in step S201 is added to the MUA solution. – Au + Solution.

4. The method for preparing a ratiometric fluorescent probe according to claim 1, characterized in that: 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.

5. The method for preparing a ratiometric fluorescent probe according to claim 1, characterized in that: 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, stir for no less than 1 hour, and after mixing evenly, transfer the mixture to a reactor lined with polytetrafluoroethylene, heat to 200°C and keep warm for 3 hours. After the reaction is completed, wait for the reactor to cool to room temperature, dialyze the solution in a dialysis bag for 4 hours for purification, and dry it in an oven at 60°C to obtain a blue carbon dot powder. Dissolve the blue carbon dot powder in distilled water to obtain a B-CDs solution with a concentration of 1 mg / mL, and place it in a refrigerator for use; S2. Preparation of gold nanoclusters S201, 92.2 mg GSH was completely dissolved in 14.9 mL water, the ultrasonic time was not less than 0.5 hour, the stirring time was not less than 0.5 hour, and then 5.1 mL of 1% HAuCl4 solution was added, and the mixed solution was reacted at room temperature for 30 minutes 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 subjected to ultrasonic treatment for no less than 0.5 hours and stirring for no less than 0.5 hours, the resulting mixed solution emitted orange-red fluorescence under ultraviolet light, and aged at room temperature for 5 hours. The aged solution was transferred to a dialysis bag for dialyzing, and dialyzed in ultrapure water for more than 24 hours, and the MUA-AuNCs solution was obtained after purification; 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, pH = 7.4 PBS buffer.

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

7. A ratio 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 ratio 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 according to claim 8 in the detection of patulin, 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 ratio fluorescence chromatography test strip, the red fluorescence of the chromatography test strip test line is quenched and the blue fluorescence is turned on.

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

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