Method for adsorbing and detecting alternariol by using responsive fluorescent probe

Abstract

The invention belongs to the technical field of food detection, and particularly relates to a method for adsorbing and detecting alternaria alternata (AOH) through a responsive fluorescent probe. According to the invention, an AOH response type fluorescent nano probe (gamma-CD-RhB (at) APBA) based on gamma-cyclodextrin (gamma-CD), rhodamine B (RhB) and 3-aminophenylboronic acid (APBA) is constructed. Based on the fluorescence intensity change caused by the responsiveness of the fluorescent probe to AOH, the method realizes high-sensitivity detection of AOH through fluorescence signal change, the detection limit is 0.0017 mg / L, the linear range is 0.0056-2.50 mg / L. Meanwhile, the AOH is dynamically adsorbed and enriched by using a borate bond, the method has dual functions of detection and adsorption, and an efficient and accurate solution is provided for rapid screening of food pollutants.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology. Specifically, this invention relates to a method for detecting Alternaria solani by adsorption of a responsive fluorescent probe. Background Technology

[0002] Alternariol (AOH) is a secondary metabolite produced by fungi of the genus *Alternaria* spp., and is widely found in moldy grains, fruits, vegetables, and their products. As a typical mycotoxin, AOH not only leads to reduced crop yields but also threatens human health through the food chain, exhibiting potential genotoxicity, carcinogenicity, and immunosuppressive effects. In recent years, with global climate change and the increasing complexity of agricultural product storage conditions, the problem of *Alternaria* fungal contamination has become increasingly prominent. Therefore, developing rapid and sensitive AOH detection technologies is of great significance for ensuring food safety and human health.

[0003] Currently, conventional methods for detecting Alternaria alternataein mainly include chromatography (such as high-performance liquid chromatography and liquid chromatography-mass spectrometry) and immunoassay (such as enzyme-linked immunosorbent assay and colloidal gold immunochromatographic lateral flow method). CN202010868486.6 discloses the simultaneous detection of six Alternaria alternataein toxins using HPLC; CN202110309428.4 discloses the simultaneous detection of seven Alternaria alternataein toxins in animal feed using ultra-high performance liquid chromatography-mass spectrometry; CN202110781704.7 discloses the detection of Alternaria alternataein toxins using colloidal gold immunochromatography. Although chromatography offers high sensitivity and accuracy, it relies on expensive instruments, complex sample pretreatment, and specialized operators, making it difficult to meet the needs of rapid on-site screening. Immunoassay is limited by high antibody preparation costs, insufficient stability, and susceptibility to matrix interference. Fluorescent probe technology has attracted much attention in the detection of environmental pollutants and biotoxins due to its high sensitivity, rapid response, and visualization potential. However, there are no reports on the detection of Alternaria alternataein using fluorescent probe technology. Summary of the Invention

[0004] To achieve rapid and accurate detection of Alternaria alternifolia, this invention, through extensive research, proposes a method for detecting Alternaria alternifolia based on the adsorption of a responsive fluorescent probe, thus completing this invention. Specifically, the technical solution of this invention is as follows: In one aspect, the present invention discloses a method for preparing a responsive fluorescent probe γ-CD-RhB@APBA, the method comprising: Preparation of γ-CD-RhB solution: Rhodamine B (RhB) solution was slowly added dropwise to γ-cyclodextrin (γ-CD) solution, stirred thoroughly in the dark, centrifuged and the supernatant was collected. The supernatant was filtered through a filter membrane to obtain γ-CD-RhB solution. Add 3-aminophenylboronic acid (APBA) solution to the γ-CD-RhB solution obtained in step (1), stir thoroughly in the dark, centrifuge to collect the supernatant, filter through a filter membrane, dialyze the filtrate through a 3000 Da dialysis bag, and freeze-dry the dialysate to obtain the product γ-CD-RhB@APBA fluorescent probe.

[0005] In one embodiment, the mass ratio of γ-CD:RhB:APBA is 1:0.15:2. The centrifugation is performed at 4000 rpm for 15 minutes. The stirring is carried out for at least 12 hours.

[0006] In one embodiment, the present invention discloses a method for preparing a responsive fluorescent probe γ-CD-RhB@APBA, the method comprising: Take 1.0 g of γ-CD powder (molecular weight 1297.0 g / mol, 25.7 mM), add it to 30.0 mL of deionized water, and stir at 50.0 ℃ until transparent; then take 0.15 g of RhB (molecular weight 479.0 g / mol, 15.7 mM), dissolve it in 20.0 mL of deionized water, sonicate in the dark, and slowly add it dropwise to the γ-CD solution. Stir at room temperature in the dark for 15.0 h to obtain the reaction solution. Centrifuge the reaction solution at 4000 rpm for 15.0 min, collect the supernatant, and filter it through a 0.22 μm filter membrane to obtain the γ-CD-RhB solution.

[0007] APBA (15.3 mM, 10.0 mL) was added to solution (1), and the mixture was stirred in the dark for 24.0 h to obtain the reaction solution. The reaction solution was centrifuged at 4000 rpm for 15.0 min, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was dialyzed through a 3000 Da dialysis bag for 6.0 h, and finally freeze-dried to obtain the product γ-CD-RhB@APBA fluorescent probe.

[0008] In one aspect, the present invention discloses a method for detecting Alternaria alterniflora by adsorption of a responsive fluorescent probe, characterized in that the method comprises the following steps: S1: The γ-CD-RhB@APBA fluorescent probe was mixed with the test sample containing Alternaria solaniol. After reacting for a period of time, the mixture was transferred to a fluorescence dish, and the fluorescence intensity Fn of the reactant at 587 nm under an excitation wavelength of 405 nm was measured using a fluorescence spectrophotometer. S2: Input the fluorescence intensity value obtained in step S1 into the linear equation constructed based on different concentrations of Alternaria solani standards to calculate the Alternaria solani concentration in the sample.

[0009] In one embodiment, the standard curve is prepared as follows: At room temperature, the γ-CD-RhB@APBA fluorescent probe and different concentration gradients of Alternaria alterniflora are added to Tris-HCl buffer, thoroughly mixed, and after reacting for a period of time, transferred to a fluorescence dish. The fluorescence intensity of the reactants at 587 nm under a 405 nm excitation wavelength is measured using a fluorescence spectrophotometer and denoted as Fo. S Then, using the fluorescence intensity Fs values ​​measured from Alternaria solani standards at different concentration gradients and the concentrations of the standards, a linear regression equation for fluorescence ΔF = XC was constructed. AOH + y, where C AOH This refers to the concentration of the standard.

[0010] In one aspect, the present invention discloses a method for detecting Alternaria alterniflora by adsorption of a responsive fluorescent probe, comprising the following steps: S1: Determination of fluorescence intensity at 587 nm for Alternaria solani standard test solutions with different concentration gradients. F: Under room temperature conditions, the γ-CD-RhB@APBA fluorescent probe and Alternaria solani at different concentration gradients were added to Tris-HCl buffer and reacted thoroughly. The mixture was incubated at 30.0 ℃ for 5.0 min, and then transferred to a fluorescence dish. The fluorescence intensity at 587 nm (excitation wavelength 405 nm) was then measured using a fluorescence spectrophotometer and recorded as F. S ; S2: Constructing a linear regression equation for the standards: Using the fluorescence intensity Fs values ​​measured from Alternaria solani standards at different concentration gradients and the concentrations of the standards, a linear regression equation for fluorescence is constructed: ΔF = XC AOH + y, where C AOH For standard concentration; S3: Take the sample to be tested, repeat step 1 to obtain the fluorescence intensity Fn of each sample, and substitute it into the corresponding linear equation to obtain the concentration of Alternaria solani in the sample to be tested.

[0011] In one embodiment: In step S1, the pH of the Tris-HCl buffer is 8.3 – 8.7; the concentration of the γ-CD-RhB@APBA fluorescent probe is 300.0 mg / L; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 30.0 ℃; and the reaction time is 5.0 min.

[0012] In one embodiment: In S2, the linear regression equation for the fluorescence spectrophotometer is ΔF = 739.16C. AOH +596.05; In one aspect, the present invention discloses the application of the responsive fluorescent probe γ-CD-RhB@APBA in the detection of Alternaria alterniflora.

[0013] In one aspect, the present invention discloses a kit for detecting Alternaria alternifolia by adsorption of a responsive fluorescent probe, the kit comprising a responsive fluorescent probe γ-CD-RhB@APBA and a buffer solution; preferably, the buffer solution is a Tris-HCl buffer solution with a pH of 8.3-8.7. Beneficial effects

[0014] This invention prepares a responsive fluorescent probe γ-CD-RhB@APBA based on a specific recognition mechanism. Fluorescence signal changes are triggered by interactions between the target analyte and probe molecules (such as hydrogen bonding, π-π stacking, or chemical bonding), enabling qualitative and quantitative analysis of Alternaria alterniflora. Compared with existing chromatographic and fluorescence detection techniques, the detection time is shorter, requiring only 5.0 minutes to observe fluorescence changes.

[0015] The detection method developed in this invention has a recovery rate of ≥91.6% in grain samples. Furthermore, through the specific coordination of boric acid-o-dihydroxyl groups and the molecular sieve effect of γ-CD cavities, it effectively eliminates the interference of the aldermophyll analog aldermophyll methyl ether. The limit of detection (LOD) is as low as 0.0017 mg / L, providing an efficient and accurate solution for rapid screening of food contaminants. Attached Figure Description

[0016] Figure 1 1. TEM images of (A)γ-CD-RhB and (B)γ-CD-RhB@APBA.

[0017] Figure 2 The infrared spectra of γ-CD-RhB and γ-CD-RhB@APBA are shown below.

[0018] Figure 3 , is the hydrodynamics of (A) γ-CD-RhB and γ-CD-RhB@APBA and (B) is the Zeta potential of γ-CD-RhB and γ-CD-RhB@APBA.

[0019] Figure 4 To verify the feasibility of detecting Alternaria solaniol (AOH) with γ-CD-RhB@APBA, fluorescence absorption diagrams and corresponding solution color illustrations were obtained for different groups (1) γ-CD-RhB, (2) γ-CD-RhB + AOH, (3) γ-CD-RhB@APBA, and (4) γ-CD-RhB@APBA + AOH.

[0020] Figure 5 Optimization of conditions for the detection of Alternaria solani (AOH) by γ-CD-RhB@APBA, including pH and temperature.

[0021] Figure 6Fluorescence spectra and standard curves of standards containing different concentrations of Alternaria solani (AOH) are shown.

[0022] Figure 7 Selectivity tests for the γ-CD-RhB@APBA detection system. 1-15 represent blank, Alternaria alterniflora (AOH), Alternaria alterniflora methyl ether (AME), and K, respectively. + Mg 2+ Co 2+ Fe 2+ Ca 2+ Na + Cu 2+ NO3 - CO3 2- Al 3+ F - SO4 2- . Detailed Implementation

[0023] Please see Figure 1-7 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and to provide a clear understanding of the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0024] Reagents used in the embodiments of this invention: The raw materials used in this invention were: Rhodamine B, γ-cyclodextrin, and 3-aminophenylboronic acid (APBA) purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Tris-HCl buffer solution (Tris-HCl) purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and AOH purchased from Qingdao Pribang Biotechnology Co., Ltd. All water used in the experiments was deionized water.

[0025] Unless otherwise specified, all reagents or materials described in the following examples are commercially available.

[0026] The technical solution of the present invention will be further described in detail below with reference to examples. Example 1: Synthesis of γ-CD-RhB@APBA fluorescent probe

[0027] A method for preparing a responsive fluorescent probe γ-CD-RhB@APBA, the method comprising: Preparation of γ-CD-RhB solution: Rhodamine B (RhB) solution was slowly added dropwise to γ-cyclodextrin (γ-CD) solution, stirred thoroughly in the dark, centrifuged and the supernatant was collected. The supernatant was filtered through a filter membrane to obtain γ-CD-RhB solution. Add 3-aminophenylboronic acid (APBA) solution to the γ-CD-RhB solution obtained in step (1), stir thoroughly in the dark, centrifuge to collect the supernatant, filter through a filter membrane, dialyze the filtrate through a 3000 Da dialysis bag, and freeze-dry the dialysate to obtain the product γ-CD-RhB@APBA fluorescent probe.

[0028] Preferably, the mass ratio of γ-CD:RhB:APBA is 1:0.15:2. The centrifugation is performed at 4000 rpm for 15 minutes. The stirring is carried out for at least 12 hours.

[0029] This embodiment discloses a method for preparing the responsive fluorescent probe γ-CD-RhB@APBA, the method comprising: Take 1.0 g of γ-CD powder (molecular weight 1297.0 g / mol, 25.7 mM), add it to 30.0 mL of deionized water, and stir at 50.0 ℃ until transparent; then take 0.15 g of RhB (molecular weight 479.0 g / mol, 15.7 mM), dissolve it in 20.0 mL of deionized water, sonicate in the dark, and slowly add it dropwise to the γ-CD solution, stirring at room temperature in the dark for 15.0 h. Centrifuge the reaction solution at 4000 rpm for 15.0 min, and filter the supernatant through a 0.22 μm filter membrane to obtain the γ-CD-RhB solution.

[0030] APBA (15.3 mM, 10.0 mL) was added to the γ-CD-RhB solution and stirred in the dark for 24.0 h. The reaction solution was centrifuged at 4000 rpm for 15.0 min, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was dialyzed through a 3000 Da dialysis bag for 6.0 h, and finally freeze-dried to obtain the product γ-CD-RhB@APBA fluorescent probe. Example 2: Method system and feasibility verification for the detection of Alternaria alterniflora (AOH) using γ-CD-RhB@APBA

[0031] The following four reaction systems were designed: (1) γ-CD-RhB, (2) γ-CD-RhB + AOH, (3) γ-CD-RhB@APBA, and (4) γ-CD-RhB@APBA + AOH. Tris-HCl was used as the buffer. After the reaction was complete, the mixture was transferred to a fluorescence dish, and the fluorescence intensity at 587 nm was measured using a fluorescence spectrophotometer. The results are shown below. Figure 4This indicates that AOH can enhance the fluorescence of γ-CD-RhB@APBA, causing a significant change in fluorescence intensity. This demonstrates the feasibility of the detection system proposed in this invention.

[0032] In the above verification steps, the concentration of the γ-CD-RhB@APBA fluorescent probe was 300.0 mg / L; the concentration of AOH was 2.5 mg / L; the pH of the added Tris-HCl buffer was 8.3 – 8.7; the reaction temperature was 30.0 ℃; and the mixing and reaction time was 5.0 min. Example 3: Optimal conditions for the detection of Alternaria alterniflora (AOH) using γ-CD-RhB@APBA

[0033] To optimize the analytical performance of γ-CD-RhB@APBA, detailed tests were conducted on the pH and temperature parameters of the detection system.

[0034] The performance of this system was investigated within a pH range of 7.0–9.5, and Tris-HCl was selected as the reaction buffer. The results are as follows: Figure 5 As shown in Figure A, when the pH value is in the range of 7.0 to 8.5, the fluorescence intensity of the reaction system at 587 nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 8.5, the fluorescence intensity decreases with increasing pH value. Therefore, 8.5 was chosen as the optimal pH value for the system.

[0035] The performance of this system was investigated in the temperature range of 25.0–45.0 °C. The results are as follows: Figure 5 As shown in Figure B, when the temperature ranges from 25.0 ℃ to 30.0 ℃, the fluorescence intensity of the reaction system at 587 nm increases with increasing reaction temperature. When the temperature exceeds 30.0 ℃ and increases to 45.0 ℃, the fluorescence intensity decreases with increasing temperature. Therefore, 30.0 ℃ was chosen as the optimal temperature for the system. Example 4: Establishment of standard curves for the detection of different concentrations of Alternaria solani (AOH) standards by γ-CD-RhB@APBA

[0036] Alternaria hydrochloride (AOH) solutions of different concentration gradients (0.05, 0.10, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, and 2.75 mg / L) were reacted with 300.0 mg / L γ-CD-RhB@APBA fluorescent probe in Tris-HCl buffer at 30.0 °C for 5.0 min. After the reaction, the reaction solution was transferred to a fluorescence dish, and the fluorescence intensity Fs at 587 nm was measured using a fluorescence spectrophotometer. The fluorescence intensity at 587 nm increased with increasing AOH concentration.

[0037] Therefore, using fluorescence intensity Fs as the ordinate and AOH concentration as the abscissa, a linear equation was fitted using analytical software: ΔF = XC AOH + y, where C AOH This refers to the concentration of the standard. For example... Figure 6 As shown, the concentration of AOH and the fluorescence intensity Fs exhibit a linear relationship within the range of 0.0056 – 2.50 mg / L, with the fluorescence intensity regression equation being ΔF = 739.16C. AOH The correlation coefficient was +596.05, the limit of detection was 0.99722, and the detection limit was 0.0017 mg / L. Furthermore, the fluorescence of the solution exhibited a color gradient (from light orange to dark orange) as the concentration of AOH increased, enabling linear detection of AOH concentration. Example 5: Selectivity test of γ-CD-RhB@APBA for the detection of AOH

[0038] To evaluate the selectivity of γ-CD-RhB@APBA for AOH, several representative substances were used as proof-of-concept, including blank, Alternaria alternifolia (AOH), Alternaria alternifolia methyl ether (AME), KCl, MgCl2, CoCl2, FeSO4, CaCO3, NaF, CuCl2, AgNO3, and AlCl3. At room temperature, the γ-CD-RhB@APBA fluorescent probe was reacted with each of these substances in Tris-HCl buffer; after thorough mixing, the reaction was carried out for 5.0 min, and then transferred to a fluorescence dish. The fluorescence intensity F at 587 nm was measured. The results are shown below. Figure 7 As shown, only the fluorescence intensity F of AOH showed a significant increase, indicating that the detection system of the present invention has good selectivity for AOH.

[0039] In the above verification steps, the concentration of the γ-CD-RhB@APBA fluorescent probe was 300.0 mg / L; the concentration of the selective verification substance was 2.5 mg / L; the pH of the added Tris-HCl buffer was 8.3 – 8.7; the reaction temperature was 30.0 ℃; and the mixing and reaction time was 5.0 min. Example 6: Validation of AOH detection in real samples using γ-CD-RhB@APBA

[0040] To verify the sensing performance of γ-CD-RhB@APBA in AOH detection in practical sample applications, grains were selected as representative samples. The grains were randomly purchased from a large supermarket in Hefei, China. Grains (100.0 mg / mL, 10.0 mL deionized water) were adjusted to pH = 8.5. The supernatant was collected after centrifugation at 10000 rpm for 10.0 min, filtered through a 0.22 mm filter membrane, and then 0.50, 1.50, and 2.50 mg / L AOH were added, respectively. At room temperature, γ-CD-RhB@APBA was reacted with the above mixed solutions in Tris-HCl buffer; after thorough mixing, the reaction was allowed to proceed for 5.0 min, and then transferred to a fluorescence dish. The fluorescence intensity Fn at 587 nm was measured. Substituting the obtained fluorescence intensity Fn into the corresponding linear equation, the AOH results measured in fluorescence detection mode are shown in Table 1.

[0041] To verify the sensing performance of γ-CD-RhB@APBA in AOH detection in practical sample applications, milk powder was selected as a representative sample. The milk powder was randomly purchased from a large supermarket in Hefei, China. Milk powder (100.0 mg / mL, 10.0 mL deionized water) was adjusted to pH = 8.5. The supernatant was collected after centrifugation at 10000 rpm for 10.0 min, filtered through a 0.22 mm filter membrane, and then 0.50, 1.50, and 2.50 mg / L AOH were added, respectively. At room temperature, γ-CD-RhB@APBA was reacted with the above mixed solutions in Tris-HCl buffer; after thorough mixing, the reaction was allowed to proceed for 5.0 min, and then transferred to a fluorescence dish. The fluorescence intensity Fn at 587 nm was measured. Substituting the obtained fluorescence intensity Fn into the corresponding linear equation, the AOH results measured in fluorescence detection mode are shown in Table 1.

[0042] To verify the sensing performance of γ-CD-RhB@APBA in AOH detection in practical sample applications, soybean powder was selected as a representative sample. The soybean powder used in this test was randomly purchased from a large supermarket in Hefei, China. Soybean powder (100.0 mg / mL, 10.0 mL deionized water) was adjusted to pH = 8.5. The supernatant was collected after centrifugation at 10000 rpm for 10.0 min, filtered through a 0.22 mm filter membrane, and then 0.50, 1.50, and 2.50 mg / L AOH were added, respectively. At room temperature, γ-CD-RhB@APBA was reacted with the above mixed solutions in Tris-HCl buffer; after thorough mixing, the reaction was allowed to proceed for 5.0 min, and then transferred to a fluorescence dish. The fluorescence intensity Fn at 587 nm was measured. Substituting the obtained fluorescence intensity Fn into the corresponding linear equation, the AOH results measured in fluorescence detection mode are shown in Table 1.

[0043] Table 1 shows the detection of AOH in actual samples by γ-CD-RhB@APBA. The actual samples were cereals, milk powder, and soy powder.

[0044] Table 1 ; The results show that the reaction system can be applied to the detection of actual samples, with a recovery rate between 91.6% and 115.8% and a relative standard deviation (RSD) of less than 2.00%.

[0045] In summary, this invention discloses a novel method for detecting Alternaria alterniflora based on the adsorption of a responsive fluorescent probe. By designing a fluorescent probe molecule with targeted recognition function and combining it with a highly efficient adsorption carrier, an integrated "enrichment-detection" sensing platform is constructed. This invention constructs an AOH-responsive fluorescent nanoprobe (γ-CD-RhB@APBA) based on γ-cyclodextrin (γ-CD), rhodamine B (RhB), and 3-aminophenylboronic acid (APBA). This probe forms a fluorescence-enhancing complex by encapsulating RhB through the hydrophobic cavity of γ-CD. Subsequently, the boric acid group of APBA binds to the hydroxyl group of γ-CD, initiating fluorescence quenching. In the presence of Alternaria alterniflora (AOH), its ortho-dihydroxyl group preferentially forms a stable five / six-membered cycloboronic ester with APBA, competitively releasing γ-CD and restoring RhB fluorescence. This method achieves highly sensitive detection of AOH through changes in fluorescence signal (detection limit 0.0017 mg / L, linear range 0.0056-2.50 mg / L), while utilizing the dynamic adsorption and enrichment of AOH through borate ester bonds, thus combining detection and adsorption functions to provide an efficient and accurate solution for rapid screening of food contaminants.

[0046] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A method for detecting Alternaria solani by adsorption of a responsive fluorescent probe, characterized in that, The method includes the following steps: S1: The γ-CD-RhB@APBA fluorescent probe was mixed with the test sample containing Alternaria solaniol. After reacting for a period of time, the mixture was transferred to a fluorescence dish, and the fluorescence intensity Fn of the reactant at 587 nm under an excitation wavelength of 405 nm was measured using a fluorescence spectrophotometer. S2: Input the fluorescence intensity value obtained in step S1 into the linear equation constructed based on different concentrations of Alternaria solani standards to calculate the Alternaria solani concentration in the sample.

2. The method for detecting Alternaria solani by adsorption of a responsive fluorescent probe according to claim 1, characterized in that, The method for constructing the linear equation in step S2 includes: (1) Under normal temperature conditions, the γ-CD-RhB@APBA fluorescent probe and different concentration gradients of Alternaria alterniflora were added to Tris-HCl buffer, stirred thoroughly, and after reacting for a period of time, the mixture was transferred to a fluorescence dish. The fluorescence intensity of the reactants at 587 nm under an excitation wavelength of 405 nm was measured using a fluorescence spectrophotometer and recorded as F. S ; (2) A fluorescence linear regression equation ΔF = XC was constructed using the fluorescence intensity Fs values ​​measured from Alternaria solani standards at different concentration gradients and the concentrations of the standards. AOH + y, where C AOH This refers to the concentration of the standard.

3. The method according to claim 2, characterized in that, The test samples or standards were dissolved in Tris-HCl buffer with a pH of 8.3-8.7; the concentration of the γ-CD-RhB@APBA fluorescent probe was 300.0 mg / L; the total volume of the mixed solution was 200.0 μL; the reaction temperature was 30.0 ℃; and the reaction time was 5.0 min.

4. The method according to claim 2, characterized in that, The fluorescence linear regression equation is ΔF = 739.16C. AOH +596.

05.

5. A method for preparing a responsive fluorescent probe γ-CD-RhB@APBA, characterized in that, The method includes: (1) Preparation of γ-CD-RhB solution: Rhodamine B (RhB) solution was slowly added dropwise to γ-cyclodextrin (γ-CD) solution, stirred thoroughly in the dark, centrifuged and the supernatant was collected. The supernatant was filtered through a filter membrane to obtain γ-CD-RhB solution. (2) Add 3-aminophenylboronic acid (APBA) solution to the γ-CD-RhB solution obtained in step (1), stir thoroughly in the dark, centrifuge to collect the supernatant, filter through a filter membrane, dialyze the filtrate through a 3000 Da dialysis bag, and freeze-dry the dialysate to obtain the product γ-CD-RhB@APBA fluorescent probe.

6. The method for preparing the responsive fluorescent probe γ-CD-RhB@APBA according to claim 5, characterized in that, The mass ratio of γ-CD:RhB:APBA is 1:0.15:2, the centrifugation is 4000 rpm for 15 min, and the stirring is sufficient for at least 12 h.

7. A method for preparing a responsive fluorescent probe γ-CD-RhB@APBA, characterized in that, The method includes: Take 1.0 g of γ-CD powder, add it to 30.0 mL of deionized water, and stir at 50.0 ℃ until transparent; then take 0.15 g of RhB, dissolve it in 20.0 mL of deionized water, sonicate it in the dark, and slowly add it dropwise to the γ-CD solution. Stir at room temperature in the dark for 15.0 h to obtain the reaction solution. Centrifuge the reaction solution at 4000 rpm for 15.0 min, take the supernatant and filter it through a 0.22 μm filter membrane to obtain the γ-CD-RhB solution. APBA was added to solution (1), and the mixture was stirred in the dark for 24.0 h to obtain a reaction solution. The reaction solution was centrifuged at 4000 rpm for 15.0 min, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was dialyzed through a 3000 Da dialysis bag for 6.0 h, and finally freeze-dried to obtain the product γ-CD-RhB@APBA fluorescent probe.

8. The application of the fluorescent probe γ-CD-RhB@APBA prepared by any one of claims 5-8 in the detection of Alternaria alternifolia.

9. A kit for detecting Alternaria solani by adsorption of a responsive fluorescent probe, characterized in that, The kit includes the responsive fluorescent probe γ-CD-RhB@APBA prepared by any one of the methods described in claims 5-8 and a buffer solution.

10. The reagent kit according to claim 9, characterized in that, The buffer solution is a Tris-HCl buffer solution with a pH of 8.3-8.7.

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