Fluorescent probe for detecting ochratoxin a and preparation method and application thereof

Abstract

The present application relates to the technical field of harmful substance detection, in particular to a fluorescent probe for detecting ochratoxin A and a preparation method and application thereof.The fluorescent probe for detecting ochratoxin A is a complex of a fluorescent dye DOCE and serum albumin combined by hydrogen bonds; wherein the chemical structural formula of the fluorescent dye DOCE is as shown.The fluorescent probe prepared by the present application is used for rapid quantitative or qualitative detection of ochratoxin A (OTA), and the detection method has an ultrafast response speed (5 seconds), high sensitivity (detection limit is 0.39 ppb), and high selectivity, and has been successfully applied to on-site rapid detection of real food samples such as flour, white wine and red wine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of harmful substance detection, and particularly relates to a fluorescent probe for detecting ochratoxin A as well as a preparation method and application thereof. BACKGROUND

[0002] Ochratoxin A (OTA) is a primary mycotoxin and secondary fungal metabolite produced by fungi (e.g., Aspergillus). It is often produced due to improper handling during the production, processing, storage and transportation of food (including cereals and their derivatives, beer, wine, spices and coffee). This contamination extends to the entire food supply chain, causing huge economic losses. Studies have shown that OTA can penetrate into the food chain of humans and animals, and has serious harmful effects on the health of both, such as carcinogenicity, nephrotoxicity, hepatotoxicity, genotoxicity and neurotoxicity. Therefore, OTA has been listed as a 2B carcinogen by the International Agency for Research on Cancer (IARC). The European Commission has established strict guidelines, stipulating that the maximum allowable residue of OTA in food is 0.5-30 ppb. Similarly, the National Food Safety Standard of China GB2761-2017 stipulates that the maximum allowable residue (MRL) of OTA in food should be in the range of 2-10 ppb. Therefore, it is of great significance to develop a reliable analysis method for OTA in food.

[0003] At present, there are many methods for detecting OTA. Traditional methods include high performance liquid chromatography (HPLC), high performance liquid chromatography-fluorescence detection (HPLC-FLD), liquid chromatography-mass spectrometry (LC-MS / MS) and high performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Although these methods have good sensitivity and accuracy, they usually require expensive instruments, professional operators and complex sample preparation processes, and thus are not suitable for easy and rapid detection of analytes in actual samples.

[0004] Compared with traditional methods, fluorescence methods have the characteristics and advantages of fast response, high sensitivity, high selectivity and direct in-situ detection of target substances. So far, many fluorescent probes for detecting OTA have been reported. Most of them are aptamer probes, which use OTA-specific aptamer as the recognition fragment. However, the reaction time of these sensing probes is usually long (> 10 minutes), which is due to the decrease in affinity to OTA caused by modification and the slow aptamer reconstitution process. Other fluorescence methods have their own shortcomings, such as poor sensitivity, poor portability, narrow application range, etc. Therefore, it is urgent to develop a method that can quickly and conveniently detect OTA in actual food samples. SUMMARY

[0005] In view of the above deficiencies of the prior art, the present application provides a fluorescent probe for detecting ochratoxin A and a preparation method and application thereof, aiming at solving the technical problems of high cost, slow detection speed, complex operation and narrow application range of the existing detection method for detecting ochratoxin A.

[0006] Specifically, the technical scheme of the present application is as follows:

[0007] The present application provides a fluorescent probe for detecting ochratoxin A, which is a complex of a fluorescent dye DOCE and serum albumin (ALB) combined by hydrogen bonds.

[0008] The chemical structural formula of the fluorescent dye DOCE is as follows:

[0009]

[0010] The present application also provides a preparation method of the fluorescent probe, comprising the following steps:

[0011] 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde are reacted to obtain an intermediate product HSF;

[0012] The intermediate product HSF and 2-ethylbutyryl chloride are subjected to esterification reaction to obtain the fluorescent dye DOCE;

[0013] The fluorescent dye DOCE is mixed with serum albumin to obtain the fluorescent probe.

[0014] The preparation method of the fluorescent probe, wherein the step of reacting 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde to obtain an intermediate product HSF comprises:

[0015] 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde are dissolved in ethanol to obtain a reaction solution, potassium hydroxide solution is added to the reaction solution, stirring is performed at room temperature, and then the reaction solution is placed in an ice bath, hydrogen peroxide solution is added, and stirring is performed at room temperature again, ice water and hydrochloric acid are added to obtain a precipitate, and the precipitate is filtered and washed to obtain the intermediate product HSF.

[0016] The preparation method of the fluorescent probe, wherein the step of dissolving the intermediate product HSF and 2-ethylbutyryl chloride in N,N-dimethylformamide to obtain a mixed solution for esterification reaction to obtain the fluorescent dye DOCE comprises:

[0017] The intermediate product HSF and 2-ethylbutyryl chloride are dissolved in N,N-dimethylformamide to obtain a mixed solution, cesium carbonate is added to the mixed solution, stirring is performed at room temperature, esterification reaction is performed, and then the fluorescent dye DOCE is extracted by dichloromethane, dried, distilled under reduced pressure and purified.

[0018] The preparation method of the fluorescent probe, wherein the molar ratio of the fluorescent dye DOCE to the serum albumin is 1:1.

[0019] The application provides a method for quantitatively detecting ochratoxin A (OTA) in flour, comprising the following steps:

[0020] The flour is soaked in a mixed solution of PBS buffer and methanol to obtain a flour mixture, the flour mixture is vortexed and ultrasonically treated to obtain a suspension, and the supernatant is taken as a test solution after centrifugation;

[0021] The fluorescent probe is added to the test solution, ochratoxin A solutions with different concentration gradients are added dropwise, the fluorescence spectrum is measured, a standard curve of the fluorescence intensity ratio of the emission peak to the concentration of ochratoxin A is established, and the detection limit of ochratoxin A is determined;

[0022] The standard curve is used to quantitatively detect the content of ochratoxin A in unknown flour samples.

[0023] The method for quantitatively detecting ochratoxin A in flour, wherein the mixing ratio of the PBS buffer to the methanol is 4:1.

[0024] The method for quantitatively detecting ochratoxin A in flour, wherein the fluorescence intensity ratio of the emission peak is the ratio of the emission peaks at 450 nm and 575 nm in the fluorescence spectrum.

[0025] The application further provides a method for qualitatively detecting ochratoxin A in grape wine, comprising the following steps:

[0026] The grape wine is filtered to obtain a filtrate from which insoluble substances and pigments are removed, the fluorescent probe is added to the filtrate to serve as a blank group, ultraviolet light irradiation is performed, and the fluorescence color of the blank group is photographed;

[0027] Ochratoxin A solution is added to the filtrate, the fluorescent probe is added to the filtrate to serve as a test group, ultraviolet light irradiation is performed, and the fluorescence color of the test group is photographed;

[0028] The fluorescence colors of the blank group and the test group are compared, and a qualitative analysis standard of ochratoxin A is established.

[0029] The qualitative analysis standard is used to qualitatively detect ochratoxin A in unknown grape wine samples.

[0030] The method for qualitatively detecting ochratoxin A in grape wine, wherein the ultraviolet light irradiation adopts an excitation wavelength of 365 nm.

[0031] Beneficial effects:

[0032] The application provides a fluorescent probe for detecting ochratoxin A and a preparation method and application thereof, the fluorescent probe is a complex of a fluorescent dye DOCE and serum albumin ALB combined through hydrogen bonds, and can be used for rapid quantitative or qualitative detection of ochratoxin A (OTA), the detection method of OTA in the application has an ultrafast response speed (5 seconds), high sensitivity (detection limit is 0.39 ppb), and high selectivity. When the fluorescent probe detects OTA, the fluorescent signal thereof realizes color change from yellow to blue, the response signal can be directly recognized by a smart phone, and has been successfully applied to on-site rapid detection of real food samples such as flour, white wine and red wine. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is a nuclear magnetic resonance hydrogen spectrum of the chemical structure of the fluorescent dye DOCE in the embodiment of the application.

[0034] Figure 2 It is a nuclear magnetic resonance carbon spectrum of the chemical structure of the fluorescent dye DOCE in the embodiment of the application.

[0035] Figure 3 It is a mass spectrum of the chemical structure of the fluorescent dye DOCE in the embodiment of the application.

[0036] Figure 4 Fig. 1 is a fluorescence spectrum of serum albumin ALB titrating the fluorescent dye DOCE (10 μM) in the embodiment of the application (a), a peak intensity Job's plot analysis of the fluorescent dye DOCE mixed with serum albumin ALB at different ratios (the total concentration is kept at 10 μM) (b), a fluorescence decay curve before and after the combination of the fluorescent dye DOCE and serum albumin ALB (IRF: instrument response function, the laser source is a NanoLED with a wavelength of 392 nm) (c), an AutoDock 4.0 molecular docking experiment result graph (the fluorescent dye DOCE is shown in black, and the serum albumin ALB is shown in gray) (d), and a two-dimensional graph of the fluorescent probe DOCE@ALB processed by Ligplot (e).

[0037] Figure 5 Fig. 2 is a chemical structure of various mycotoxins (a), a binding mode and binding energy of each mycotoxin and human serum albumin ALB (protein database: 4K2C) calculated by using a molecular docking technology (AutoDock 4.0) (b), a chemical structure graph of the fluorescent dye DOCE and the fluorescent probe (c), and a schematic diagram of an IDA sensing mechanism of the fluorescent probe DOCE@ALB for selectively detecting OTA (d).

[0038] Figure 6The sensing performance test chart of the fluorescent probe DOCE@ALB on ochratoxin A (OTA) in the embodiment of the present application, which includes: (a) fluorescence response time, (b) spectral response mode, (c) linear response curve and detection limit, (d) detection specificity.

[0039] Figure 7 The sensing performance test chart of the fluorescent probe DOCE@ALB on OTA in flour extract in the embodiment of the present application, which includes: (a) extraction steps of OTA in flour, (b) fluorescence spectral response of the probe to OTA, (c) linear relationship between fluorescence intensity ratio and OTA concentration.

[0040] Figure 8 The rapid qualitative detection result chart of the fluorescent probe DOCE@ALB on OTA in wine in the embodiment of the present application, which includes: (a) white wine, (b) red wine; C in the chart is a blank group (without OTA), and T is a test group (with OTA). DETAILED DESCRIPTION

[0041] The present application provides a fluorescent probe for detecting ochratoxin A and a preparation method and application thereof, so as to make the purpose, technical scheme and effect of the present application more clear and explicit. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the protection scope of the present application.

[0042] The embodiment of the present application provides a fluorescent probe for detecting ochratoxin A, and the fluorescent probe is a complex of a fluorescent dye DOCE and serum albumin (ALB) combined by hydrogen bonds.

[0043] The chemical structural formula of the fluorescent dye DOCE is as follows:

[0044]

[0045] The fluorescent dye DOCE molecule is combined with the drug site (DS1) of serum albumin ALB by hydrogen bonds to form a complex DOCE@ALB. After the fluorescent dye DOCE is combined with serum albumin ALB, the intramolecular rotation of the fluorescent dye DOCE is limited, and the fluorescence lifetime is prolonged. The serum albumin ALB is a protein rich in serum, has a variety of topological binding cavities, and can be combined with a variety of ligands, such as amino acids, lipids, drugs and small molecule fluorescent dyes. Therefore, the serum albumin ALB can be combined with the small molecule fluorescent dye DOCE by hydrogen bonds to construct a host-guest supramolecular system, which can be used as a fluorescent probe for detecting ochratoxin A.

[0046] The embodiment of the present application provides a preparation method of a fluorescent probe, which comprises the following steps:

[0047] reacting 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde to obtain an intermediate product HSF;

[0048] esterifying the intermediate product HSF and 2-ethylbutyryl chloride to obtain the fluorescent dye DOCE;

[0049] mixing the fluorescent dye DOCE and serum albumin ALB to obtain the fluorescent probe.

[0050] The preparation method synthesizes a novel fluorescent dye DOCE through a series of chemical reactions such as Claisen-Schmidt condensation reaction, Algar-Flynn-Oyamada reaction and esterification reaction, and the full name of the fluorescent dye DOCE is 2-(4-(dimethylamino-)styryl)-4-oxy-4H-chromen-3-2-ethylbutyrate (DOCE).

[0051] In some embodiments, the step of reacting 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde to obtain an intermediate product HSF comprises:

[0052] dissolving 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde in ethanol to obtain a reaction solution, adding a potassium hydroxide solution to the reaction solution, stirring at room temperature, placing in an ice bath, adding a hydrogen peroxide solution, and then stirring at room temperature, adding ice water and hydrochloric acid to obtain a precipitate, filtering and washing the precipitate to obtain the intermediate product HSF.

[0053] In some embodiments, the step of esterifying the intermediate product HSF and 2-ethylbutyryl chloride to obtain the fluorescent dye DOCE comprises:

[0054] dissolving the intermediate product HSF and 2-ethylbutyryl chloride in N,N-dimethylformamide to obtain a mixed solution, adding cesium carbonate to the mixed solution, stirring at room temperature, performing esterification reaction, extracting with dichloromethane, drying, reducing pressure distillation and column chromatography purification to obtain the fluorescent dye DOCE.

[0055] In some embodiments, the molar ratio of the fluorescent dye DOCE to the serum albumin ALB is 1:1.

[0056] The method for quantitatively detecting ochratoxin A in flour provided by the embodiments of the present application comprises:

[0057] The flour is soaked in a mixed solution of PBS buffer and methanol to obtain a flour mixture, the flour mixture is vortexed and ultrasonically treated to obtain a suspension, and the supernatant is taken as a test solution after centrifugation;

[0058] The fluorescent probe is added to the test solution, and a solution of ochratoxin A with different concentration gradients is added dropwise, the fluorescence spectrum is measured, a standard curve of the fluorescence intensity ratio of the emission peak to the concentration of ochratoxin A is established, and the detection limit of ochratoxin A is determined;

[0059] The standard curve is used to quantitatively detect the content of ochratoxin A in an unknown flour sample.

[0060] In some embodiments, the mixing ratio of the PBS buffer and the methanol is 4:1.

[0061] In some embodiments, the fluorescence intensity ratio of the emission peak is the ratio of the emission peaks at 450 nm and 575 nm in the fluorescence spectrum.

[0062] The embodiment of the present application also provides a method for qualitatively detecting ochratoxin A in wine, comprising:

[0063] The wine is filtered to obtain a filtrate in which insoluble substances and pigments are removed, the fluorescent probe is added to the filtrate as a blank group, ultraviolet light irradiation is performed, and the fluorescence color of the blank group is photographed;

[0064] Ochratoxin A solution is added to the filtrate, and the fluorescent probe is added to the filtrate as a test group, ultraviolet light irradiation is performed, and the fluorescence color of the test group is photographed;

[0065] The fluorescence colors of the blank group and the test group are compared, and a qualitative analysis standard of ochratoxin A is established;

[0066] The qualitative detection of ochratoxin A in an unknown wine sample is performed by using the qualitative analysis standard.

[0067] In the above-mentioned methods for quantitatively or qualitatively detecting ochratoxin A, the fluorescent probe DOCEALB is used, which is a host-guest interaction supramolecular recognition system, and the signal response is generated through a competitive binding process between the target molecule and the optical indicator, which is called indicator displacement assay (IDA). The remarkable feature of IDA detection is the ultrafast response time (in seconds) for the target molecule, because it is sensitive to the change of non-covalent bond, and is not based on the change of covalent bond in the chemical sensor based on reaction. Therefore, the detection method in the embodiment of the present application has the advantages of rapidness and sensitivity.

[0068] The binding energy of ochratoxin A (OTA) to serum albumin ALB is -10.35 kcal / mol, while the binding energy of other common mycotoxins, such as aflatoxin B1 (AFB1), zearalenone (ZEN), citrinin, deoxynivalenol (DON) and patulin, to serum albumin ALB is -7.88 to -5.35 kcal / mol. Therefore, a fluorescent dye DOCE is screened, which has a binding energy of -8.59 kcal / mol to serum albumin ALB, indicating that the binding ability of the fluorescent dye DOCE to serum albumin ALB is less than that of OTA, but greater than that of other mycotoxins. Therefore, the fluorescent dye DOCE is combined with serum albumin ALB to prepare a supramolecular fluorescent probe DOCE@ALB, and OTA can destroy the binding between the fluorescent dye DOCE and serum albumin ALB, thereby causing the fluorescence response of the fluorescent probe DOCE@ALB, while other mycotoxins do not cause the fluorescence signal response. Therefore, when the fluorescent probe DOCE@ALB is applied to the qualitative and quantitative detection of ochratoxin A in food, the effect of high selectivity can be achieved.

[0069] In some specific embodiments, the ultraviolet light irradiation uses an excitation wavelength of 365 nm.

[0070] The scheme of the present application is further described below with specific examples.

[0071] Example 1

[0072] The synthetic route of the fluorescent dye DOCE is as follows:

[0073]

[0074] According to the above synthetic route, the specific preparation steps are as follows:

[0075] Dissolve 2-hydroxyacetophenone (10 mmol) and 4-(dimethylamino)cinnamaldehyde (10 mmol) in 20 mL of ethanol. Then, add 10 mL (50 mmol / L) of aqueous potassium hydroxide solution to the reaction solution, and stir at room temperature for 12 hours. Place the reactor in an ice bath, slowly add 5 mL of 30% hydrogen peroxide solution, and stir at room temperature for 12 hours. Pour the mixture into ice water, and neutralize with dilute hydrochloric acid. Collect the precipitate by filtration, and wash with cold ethanol. The intermediate product HSF is obtained in a yield of 60%. Then, dissolve 10 mmol of the intermediate product HSF and 10 mmol of 2-ethylbutyryl chloride in 20 mL of super-dry N,N-dimethylformamide. Add 5 mmol of cesium carbonate to the solution, and stir at room temperature for 12 hours. After the reaction is completed, add 100 mL of water. Extract three times with 20 mL of dichloromethane, dry over anhydrous sodium sulfate, distill under reduced pressure, and purify by column chromatography. The final product, fluorescent dye DOCE, is obtained in a yield of 60%.

[0076] Structural characterization: The proton nuclear magnetic resonance spectrum of the fluorescent dye DOCE is shown in FIG. 1, the carbon nuclear magnetic resonance spectrum of the fluorescent dye DOCE is shown in FIG. 2, and the mass spectrum of the fluorescent dye DOCE is shown in FIG. 3. These results indicate that the fluorescent dye DOCE is synthesized through a series of chemical reactions such as Claisen-Schmidt condensation, Algar-Flynn-Oyamada reaction, and esterification. Figure 1 Figure 2 Figure 3

[0077] Example 2

[0078] The fluorescent dye DOCE is combined with recombinant human serum albumin ALB (purchased from Sigma-Aldrich, item number A9731) at a molar ratio of 1:1 to prepare a supramolecular fluorescent probe DOCE@ALB. The fluorescence spectra of the fluorescent dye DOCE before and after binding to the serum albumin ALB change, and the binding mechanism and fluorescence spectrum change are as follows: As shown in FIG. 4(a), the fluorescence intensity of the fluorescent dye DOCE in water is the smallest, but after the addition of the serum albumin ALB, the fluorescence gradually increases, and an emission peak appears at 575 nm. As shown in FIG. 4(b), the Job’s plot experiment clearly indicates that the fluorescent dye DOCE and the serum albumin ALB can form a binding complex at a molar ratio of 1:1. In addition, after interacting with ALB, the fluorescence lifetime of the fluorescent dye DOCE is extended from 1.29 ns to 1.93 ns, indicating that the binding with ALB restricts the intramolecular rotation (RIR) of the fluorescent dye DOCE, as shown in FIG. 4(c). The possible binding mode of the fluorescent probe DOCE@ALB is calculated by molecular docking technology, as shown in FIG. 4(d). Figure 4 Figure 4 Figure 4 Figure 4 ​​​​​​As shown in (d) and (e). The results indicate that the fluorescent dye DOCE binds to the drug site (DS1) of albumin via hydrogen bonds.

[0079] The fluorescent probe DOCE@ALB solution should be stored in a dark and low-temperature environment (below 10 degrees Celsius).

[0080] Mechanism of action of the fluorescent probe DOCE@ALB for rapid and highly selective detection of ochratoxin A (OTA)

[0081] like Figure 5 As shown in (a), when food is contaminated with mold, OTA is produced simultaneously with various mycotoxins, such as aflatoxin B1 (AFB1), zearalenone (ZEN), citrinin, deoxynivalenol (DON), and patulin. Studies have shown that all of these mycotoxins can bind to drug site 1 (DS1) on serum albumin (ALB). Figure 5 As shown in (b), the binding energy between the fungal toxin and ALB was calculated using molecular docking technology, with values ​​ranging from -10.35 to -5.35 kcal / mol. It can be seen that OTA has the highest binding affinity to ALB. Based on the IDA principle, it is assumed that by using a suitable fluorescent indicator whose binding energy to serum albumin ALB falls precisely between that of OTA and other fungal toxins, only OTA can effectively displace the indicator from its ALB complex into a free molecule. This displacement process triggers a fluorescence change in the indicator, thus allowing for selective detection of OTA. Figure 5 As shown in (c), a fluorescent indicator, 2-(4-(dimethylamino)styryl)-4-oxo-4H-chromen-3-yl-2-ethylbutyrate (DOCE), was screened. The binding energy of this fluorescent dye DOCE to serum albumin (ALB) (-8.59 kcal / mol) is between that of OTA and other mycotoxins. Theoretically, this localization allows OTA to effectively replace DOCE from its ALB-bound complex, while other mycotoxins cannot, thus achieving highly selective detection of OTA, such as... Figure 5 As shown in (d).

[0082] Example 3

[0083] Preparation of OTA standards with different concentration gradients

[0084] like Figure 6As shown in (a), the fluorescent probe DOCE@ALB itself has good light stability under ultraviolet light irradiation. After adding OTA, the fluorescent probe DOCE@ALB rapidly responds and reaches a peak within 5 seconds, and the signal stable output time is greater than 10 minutes. As shown in (a), Figure 6 As shown in (b), under 400 nm excitation light, with the increase of the concentration of OTA, the fluorescence peak of the probe at 575 nm continuously decreases, the fluorescence peak at 450 nm continuously increases, and the fluorescence color gradually changes from yellow to blue. As shown in (b), Figure 6 As shown in (c), the fluorescence intensity ratio (I450 / I575) of the fluorescent probe DOCE@ALB at 450 nm and 575 nm has a good linear relationship with the concentration of OTA, and the detection limit (LOD) calculated is 0.99 nM (about 0.39 ppb). As shown in (c), Figure 6 As shown in (d), the fluorescence response of the fluorescent probe DOCE@ALB to OTA is much higher than that of other common mycotoxins such as aflatoxin B1 (AFB1), zearalenone (ZEN), citrinin, deoxynivalenol (DON) and patulin, which proves that the fluorescent probe DOCE@ALB has good detection specificity.

[0085] Example 4

[0086] Application of the fluorescent probe DOCE@ALB in rapid quantitative detection of OTA in flour

[0087] As shown in (a), the extraction step of the flour sample extract is divided into 5 steps: Figure 7

[0088] (1) Take 5 grams of flour and immerse it in a mixed solution of 50 milliliters of PBS buffer and methanol (molar ratio of 4:1) to form a flour mixture;

[0089] (2) Vortex the flour mixture for 10 minutes;

[0090] (3) Continue ultrasonic treatment for 10 minutes to obtain a suspension;

[0091] (4) Centrifuge the obtained suspension at a speed of 7000 revolutions per minute for 10 minutes;

[0092] (5) Take the supernatant, i.e. the flour sample extract, as the test solution.

[0093] Then, 2 mL of the test solution is added to a quartz cuvette, 2 μL of the fluorescent probe DOCE@ALB with a concentration of 1 mmol / L is added by using a pipette, and finally different concentration gradients of OTA solution are added dropwise. Under 400 nm excitation light, the fluorescence spectrum is measured by a fluorescence spectrophotometer. ​

[0094] The standard curve of fluorescence intensity ratio and OTA concentration was established by the ratio of emission peaks at 450 nm and 575 nm in fluorescence spectrum, and the detection limit of OTA was determined.

[0095] As shown in Figure 7 In this embodiment, the sample extract containing different concentrations of OTA (10 nmol / L-1 μmol / L) was added, and the fluorescence spectrum of the fluorescent probe DOCE@ALB responded quickly. The fluorescence peak of the probe at 575 nm decreased, the fluorescence peak at 450 nm increased, and the fluorescence color gradually changed from yellow to blue. As shown in Figure 7 As shown in (c), the fluorescence intensity ratio (I450 / I575) of the fluorescent probe DOCE@ALB at 450 nm and 575 nm showed a good linear relationship with the concentration of OTA. The standard curve of fluorescence intensity ratio and OTA concentration was established, and the detection limit of OTA was determined. The detection limit was 3.7 ppb. Based on the curve, the OTA content in unknown flour samples can be quickly detected.

[0096] Example 5

[0097] Supramolecular fluorescent probe DOCE@ALB for rapid qualitative analysis of OTA in wine

[0098] As shown in Figure 7 The detection methods of white wine and red wine are consistent, and the processing steps are as follows:

[0099] 5 mL of wine was slowly filtered through a syringe filter column of polyacrylamide (PMA) to remove insoluble substances and pigments in the wine, and a filtrate was obtained.

[0100] Blank group: 2 mL of filtrate was added to a quartz cuvette, and 2 μL of fluorescent probe DOCE@ALB with a concentration of 1 mmol / L was added by a pipette gun as a blank control. A handheld ultraviolet lamp (365 nm excitation wavelength) was used for irradiation, and the color of the blank group solution in the cuvette was photographed.

[0101] Control group: 2 mL of filtrate with added OTA was added to a quartz cuvette, and 2 μL of fluorescent probe DOCE@ALB with a concentration of 1 mmol / L was added by a pipette gun as a test group. A handheld ultraviolet lamp (365 nm excitation wavelength) was used for irradiation, and the color of the test group solution in the cuvette was photographed. As shown in Figure 7 The fluorescence color of the blank group was yellow, and the fluorescence color of the test group was blue. By using the difference in fluorescence color between the blank group and the test group, a standard (RGB) for qualitative analysis of OTA was established. Through color comparison, the OTA in the wine sample can be qualitatively detected.

[0102] It is to be understood that the application is not limited to the examples described above, which can be modified or adapted in several ways by those skilled in the art without departing from the scope of the present application, as defined by the appended claims.

Claims

1. A fluorescent probe for detecting ochratoxin A, characterized in that, The fluorescent probe is a complex of fluorescent dye DOCE and serum albumin combined by hydrogen bond; The chemical structural formula of the fluorescent dye DOCE is as follows: The serum albumin is albumin.

2. A method for preparing the fluorescent probe according to claim 1, characterized by, The method comprises the steps of: reacting 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde to obtain an intermediate product HSF; esterifying the intermediate product HSF and 2-ethylbutyryl chloride to obtain the fluorescent dye DOCE; mixing the fluorescent dye DOCE and serum albumin to obtain the fluorescent probe.

3. The method for preparing the fluorescent probe according to claim 2, characterized in that, The step of reacting 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde to obtain an intermediate product HSF comprises: dissolving 2-hydroxyacetophenone and 4-(dimethylamino)cinnamaldehyde in ethanol to obtain a reaction solution, adding potassium hydroxide solution to the reaction solution, stirring at room temperature, placing in an ice bath, adding hydrogen peroxide solution, and then stirring at room temperature, adding ice water and hydrochloric acid to obtain a precipitate, filtering and washing the precipitate to obtain the intermediate product HSF.

4. The method for preparing the fluorescent probe according to claim 2, characterized in that, The step of dissolving the intermediate product HSF and 2-ethylbutyryl chloride in N,N-dimethylformamide to obtain a mixed solution for esterification to obtain the fluorescent dye DOCE comprises: dissolving the intermediate product HSF and 2-ethylbutyryl chloride in N,N-dimethylformamide to obtain a mixed solution, adding cesium carbonate to the mixed solution, stirring at room temperature, performing esterification, extracting with dichloromethane, drying, reducing pressure distillation, and column chromatography purification to obtain the fluorescent dye DOCE.

5. The method for preparing the fluorescent probe according to claim 2, characterized in that, The molar ratio of the fluorescent dye DOCE to the serum albumin is 1:

1.

6. A method for the quantitative detection of ochratoxin A in flour, characterized in that, The method comprises the steps of: immersing flour in a mixed solution of PBS buffer and methanol to obtain a flour mixture, vortexing and ultrasonic treating the flour mixture to obtain a suspension, and taking the supernatant as a test solution after centrifugation; adding the fluorescent probe of claim 1 to the test solution, adding ochratoxin A solution with different concentration gradients dropwise, measuring a fluorescence spectrum, establishing a standard curve of a fluorescence intensity ratio of an emission peak to an ochratoxin A concentration, and determining a detection limit of ochratoxin A; quantitatively detecting the content of ochratoxin A in an unknown flour sample by using the standard curve.

7. The method for quantitative detection of ochratoxin A in flour according to claim 6, characterized by that, The mixing ratio of the PBS buffer to the methanol is 4:

1.

8. The method for quantitative detection of ochratoxin A in flour according to claim 6, characterized by that, The fluorescence intensity ratio of the emission peak is the ratio of the emission peaks at 450 nm and 575 nm in the fluorescence spectrum.

9. A method for qualitatively detecting ochratoxin A in wine, characterized in that, The method comprises the steps of: filtering grape wine to obtain a filtrate with insoluble substances and pigments removed, adding the fluorescent probe of claim 1 to the filtrate as a blank group, performing ultraviolet irradiation, and taking a fluorescence color of the blank group; adding ochratoxin A solution to the filtrate, adding the fluorescent probe as a test group, performing ultraviolet irradiation, and taking a fluorescence color of the test group; comparing the fluorescence colors of the blank group and the test group to establish a qualitative analysis standard of ochratoxin A; qualitatively detecting ochratoxin A in an unknown grape wine sample by using the qualitative analysis standard.

10. The method for qualitatively detecting ochratoxin A in wine according to claim 9, characterized in that, The ultraviolet irradiation adopts an excitation wavelength of 365 nm.

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