Aflatoxin B1 rapid detection method based on fluorescence probe self-assembly

Abstract

The application provides a perylene imide derivative / sodium dodecyl sulfonate assembly, wherein the perylene imide derivative has a structure shown in formula (I), and the sodium dodecyl sulfonate has a structure shown in formula (II). The assembly has good water solubility, and can specifically detect aflatoxin B1.

Description

Technical Field

[0001] This invention relates to the field of biosensing and detection technology, specifically to a rapid fluorescence detection method for aflatoxin B1 in a perylene imide derivative / sodium dodecyl sulfonate assembly. Background Technology

[0002] Aflatoxin B1 is a secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus. Studies have shown that its toxicity includes acute poisoning from high-dose exposure in a short period and chronic poisoning from long-term exposure; a concentration greater than 1.0 mg / kg can produce highly toxic effects. It is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). To ensure food safety, developing accurate methods for detecting aflatoxin B1 levels in food is particularly important.

[0003] Traditional detection methods (such as liquid chromatography and enzyme-linked immunosorbent assay) rely on expensive instruments and specialized laboratory researchers, and are time-consuming, which limits the development of aflatoxin B1 detection using traditional methods. Therefore, rapid and accurate detection of aflatoxin B1 in food samples, such as wheat, is of great significance for the prevention of persistent organic pollutants and the assurance of food safety. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid fluorescent detection method for aflatoxin B1 in a perylene imide derivative / sodium dodecyl sulfonate assembly.

[0005] This invention provides a perylene imide derivative / sodium dodecyl sulfonate assembly having the structures shown in formulas (I) and (II):

[0006]

[0007] The perylene imide derivative / sodium dodecyl sulfonate assembly with the structures shown in formulas (I) and (II) above has good water solubility and can achieve highly sensitive and specific detection of aflatoxin B1, exhibiting obvious specificity and anti-interference ability in the detection.

[0008] In addition, the perylene imide derivative with the structure shown in formula (I) provided by the present invention is a small molecule cationic probe containing a perylene ring structure with good optical activity and a side chain that can ensure good solubility.

[0009] The compounds with the structure shown in the above perylene imide derivative / sodium dodecyl sulfonate assembly exhibit different conformations and aggregation states in solution in response to external stimuli, thereby causing changes in their optical properties, such as the on or quenching of solution fluorescence.

[0010] Therefore, when the above-mentioned perylene imide derivative / sodium dodecyl sulfonate assembly is used for the detection of aflatoxin B1, it can achieve a fluorescence response effect, that is, the fluorescence of the perylene imide derivative / sodium dodecyl sulfonate assembly is quenched, and this quenching effect is visible to the naked eye under 365nm ultraviolet light irradiation.

[0011] Furthermore, the detection limit of the above perylene imide derivative / sodium dodecyl sulfonate assembly is as low as 0.74 ng / mL, and its visualization effect in solution is obvious.

[0012] The excellent aflatoxin B1 detection performance of the perylene imide derivative / sodium dodecyl sulfonate assembly provided by this invention may be based on the following principle: the perylene imide derivative / sodium dodecyl sulfonate assembly of this invention can be recognized by aflatoxin B1 through non-covalent interactions (such as electrostatic attraction, hydrophobic interaction, hydrogen bonding, etc.). In aqueous solution, the two form a stable supramolecular aggregate, which leads to a change in the probe fluorescence signal.

[0013] The present invention does not have any particular limitation on the source of the above perylene imide derivative / sodium dodecyl sulfonate assembly, which can be commercially available or prepared according to methods known to those skilled in the art.

[0014] To obtain higher quality and yield perylene imide derivative / sodium dodecyl sulfonate assemblies, the present invention provides a preferred preparation method as described below for preparing perylene imide derivative / sodium dodecyl sulfonate assemblies, specifically:

[0015] This invention provides a method for preparing the above-mentioned perylene imide derivative / sodium dodecyl sulfonate assembly, comprising the following steps:

[0016] S1) The compound shown in formula (Ⅲ) is mixed with pyridine to carry out the first reaction, and the compound shown in formula (Ⅳ) is obtained;

[0017]

[0018] S2) The compound shown in formula (Ⅳ) and the compound shown in formula (Ⅴ) are reacted in the presence of imidazole to obtain the compound shown in formula (I);

[0019]

[0020] S3) The compound shown in formula (I) is mixed with the compound shown in formula (II) to carry out a third reaction, yielding a perylene imide derivative / sodium dodecyl sulfonate assembly.

[0021]

[0022] The present invention does not impose any particular limitation on the source of the compound shown in formula (V) above. For example, it can be obtained commercially or synthesized using methods provided by existing technology.

[0023] Preferably, the temperature of the first reaction is 70-80℃, more preferably 70℃; and the reaction time is 4-6h, more preferably 4h.

[0024] Preferably, the temperature of the second reaction is 90-120°C, more preferably 90°C; and the reaction time is 8-10h, more preferably 8h.

[0025] Preferably, after the second reaction is completed, the system undergoes post-processing:

[0026] The resulting reaction mixture was subjected to solvent removal and precipitation treatment in sequence to obtain the compound shown in formula (I).

[0027] Preferably, the precipitation treatment of the present invention specifically includes: precipitating the product with hydrochloric acid, centrifuging, washing the precipitated product with water, and then vacuum drying.

[0028] Preferably, the molar ratio of formula (I) to formula (II) in the third reaction is 20:1.

[0029] This invention provides the application of the above-mentioned perylene imide derivative / sodium dodecyl sulfonate assembly in the detection of aflatoxin B1.

[0030] The present invention also provides a method for detecting aflatoxin B1, using a perylene imide derivative / sodium dodecyl sulfonate assembly as a probe.

[0031]

[0032] Preferably, the concentration of the perylene imide derivative is 1 μmol / L and the concentration of sodium dodecyl sulfonate is 0.05 μmol / L.

[0033] In some embodiments of the present invention, the specific detection method is as follows: Aflatoxin B1 detection spectral testing is performed in a 10 mmol / L pH 7.0 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer solution, with the concentration of the perylene imide derivative being 1 μmol / L and the concentration of sodium dodecyl sulfate being 0.05 μmol / L. The excitation wavelength is 495 nm, and the slit width for both excitation and emission light is 3.0 nm. The fluorescence intensity at the emission wavelength of 547 nm is used to calculate the degree of fluorescence quenching. A detection standard curve is established with the concentration of aflatoxin B1 as the abscissa and the degree of fluorescence quenching as the ordinate.

[0034] Experimental results show that the detection method provided by this invention has high detection efficiency and sensitivity.

[0035] The present invention also provides a reagent, test strip or kit for detecting aflatoxin B1, comprising the perylene imide derivative / sodium dodecyl sulfonate assembly shown in formula (I) and formula (II);

[0036]

[0037] Compared with the prior art, the present invention provides a perylene imide derivative / sodium dodecyl sulfonate assembly having the structures shown in formulas (I) and (II). The above-mentioned perylene imide derivative / sodium dodecyl sulfonate assembly has good water solubility and can specifically detect aflatoxin B1. Attached Figure Description

[0038] Figure 1 The fluorescence quenching degree of perylene imide derivative / sodium dodecyl sulfonate assembly probe after adding different concentrations of aflatoxin B1 to HEPES buffer (10 mmol / L, pH=7.0);

[0039] Figure 2 The relative fluorescence intensity (λ) of the perylene imide derivative / sodium dodecyl sulfonate assembly in HEPES buffer (10 mmol / L, pH = 7.0) interacting with different substances is given. ex =495nm, λ em =547nm) Detailed Implementation

[0040] To further illustrate the present invention, the rapid fluorescence detection method for aflatoxin B1 based on perylene imide derivative / sodium dodecyl sulfonate assembly provided by the present invention will be described in detail below with reference to embodiments.

[0041] Preparation of spare materials:

[0042] Preparation of buffer solution: Weigh N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) solid and prepare 100 mL of 10 mmol / L HEPES buffer solution with distilled water. Adjust the pH to 7.0 with 1 mmol / L sodium hydroxide standard solution. Store at 4°C for later use.

[0043] Preparation of probe stock solutions: The perylene imide derivatives and sodium dodecyl sulfonate shown in formulas (I) and (II) were separately prepared into stock solutions with a concentration of 1 mmol / L using distilled water. These stock solutions were then dispensed into equal volumes into vials for later use. Before spectral testing, the stock solutions were ultrasonically mixed to carry out the third reaction. During spectral testing, the solutions were diluted to a specific concentration using prepared HEPES buffer solution (10 mmol / L, pH = 7.0) for testing.

[0044] Preparation of analytes and interfering substances: Aflatoxin B1 was prepared into a stock solution with a concentration of 1 mg / mL using dimethyl sulfoxide (DMSO). Other interfering substances were prepared into stock solutions with a concentration of 1 mg / mL using distilled water. These solutions were stored at 4°C for later use.

[0045] Preparation of actual samples: Grain samples were purchased from a local supermarket, extracted by ultrasound, centrifuged, filtered through a 0.22μm microporous membrane, and the filtrate was used for experiments.

[0046] Example 1

[0047] Preparation of the compound shown in formula (I):

[0048] 1.23 g of 2-bromoethylamine hydrobromide and 2 mL of pyridine were added to a Schlenk tube and stirred at 70 °C for 4 hours to obtain the compound shown in formula (III). Then, 3.0 g of imidazole was added, and the solution was heated at 95 °C to become a clear, dark yellow liquid. 550.3 mg of 3,4,9,10-perylenetetracarboxylic acid dianhydride was added, and the reaction was carried out at 90 °C for 8 hours. After cooling to room temperature, the product was transferred to a centrifuge tube, and the pH of the solution was lowered with 2.0 mol / L HCl solution until no more precipitate was formed. The solution was then centrifuged for 10 min (1000 r / min) to remove the supernatant. The solution was washed three times with ultrapure water and dried under vacuum to obtain the compound shown in formula (I).

[0049] Its characteristics are as follows: 1 H-NMR(CF3COOD,500MHz,ppm):5.06-4.93(2H,d),4.91-4.81(2H,d),4.81-4.70(2 H,d),4.61-4.51(1H,m),4.27-3.87(2H,m),1.18-1.08(2H,m),1.05-0.91(2H,m). 13C-NMR (125MHz, CF3COOD, ppm): δ165.71,146.68,144.80,136.57,133.30,129.53,128.88,126.57,124.57,121.44,59.72,41.07.HRMS: Exact mass calcd for C 38 H 26 N4O4 2+ :602.1943.Found:602.1941.

[0050] The compound shown in formula (I) is well soluble in water. The compound shown in formula (I) and the compound shown in formula (II) were ultrasonically reacted at 25°C for 30 min (ratio 20:1) to obtain a perylene imide derivative / sodium dodecyl sulfonate assembly (referred to as probe or assembly).

[0051] Example 2

[0052] Fluorescence spectroscopy testing:

[0053] Take 1 μL of the probe stock solution and 950 μL of HEPES buffer (10 mmol / L) and add it to a 1 mL sample cell. After mixing thoroughly, measure the fluorescence spectrum of the probe buffer and record the fluorescence intensity at 547 nm as I0. Subsequently, gradually add aflatoxin B1 at a certain concentration gradient to the sample cell, mix thoroughly, measure the corresponding fluorescence spectrum, and record the fluorescence intensity at 547 nm as I.

[0054] The results are as follows Figure 1 As shown (that is, Figure 1 This indicates that the fluorescence of the aforementioned assembly at 547 nm in HEPES (10 mmol / L, pH = 7.0) buffer is quenched by the aflatoxin B1 concentration, I / I0. ex =495nm, the slit width for excitation and emission light is 3.0nm.

[0055] The aforementioned assembly was excited at an excitation wavelength of 495 nm, emitting a maximum emission peak at 547 nm. With the addition of aflatoxin B1, the fluorescence emission intensity of the assembly gradually decreased. When the concentration of aflatoxin B1 was increased to 10 μg / mL, the quenching degree of the assembly reached 96%. Based on the method for calculating the detection limit, the detection limit of the assembly for aflatoxin B1 is 0.74 ng / mL.

[0056] Example 3

[0057] Selective studies:

[0058] Select environmental samples containing aflatoxin B1, such as cereal flour, for potential interfering substances, including common anions, cations, amino acids, and other fungal toxins: K + Ca 2+ Na + Mg 2+ Al 3+ Zn 2+ Fe 3+ The analytes included glucose, lactose, aspartic acid, tryptophan, phenylalanine, alanine, histidine, lysine, vomitoxin, zearalenone, and fumonisin B1. The concentrations of the perylene imide derivatives were 1 μmol / L, sodium dodecyl sulfate was 0.05 μmol / L, and all interfering substances were 5 μg / mL. Fluorescence spectroscopy was performed under the same conditions. The absorbance at 547 nm of the assemblies before and after the addition of interfering substances was compared, with I0 / I serving as a parameter to measure the influence of the assemblies on the analytes.

[0059] The results are as follows Figure 2 As shown (that is, Figure 2 The graph shows the relative absorbance values ​​of the aforementioned assembly interacting with aflatoxin B1 and other interfering agents in HEPES (5 mmol / L, pH 7.0) buffer.

[0060] As can be seen from the figure, except for aflatoxin B1, the I0 / I≈1 of all other substances is much lower than that of aflatoxin B1. This result indicates that the aforementioned assembly has excellent selectivity for aflatoxin B1.

[0061] Example 4

[0062] To more intuitively observe the effect of the aforementioned assembly interacting with aflatoxin B1 and its interfering substances, the changes in solution fluorescence during the selective study of aflatoxin B1 in aqueous solution were monitored under 365 nm ultraviolet light irradiation.

[0063] Choose aflatoxin B1 and the aforementioned interfering substances, such as Figure 2 As shown in Figure B, when an interfering agent at a concentration of 5 μg / mL was added to the aforementioned assembly, the fluorescence of the solution did not change, but when aflatoxin B1 was added, the fluorescence of the solution was strongly quenched.

[0064] Example 5

[0065] Application to real samples: To verify the feasibility of the aforementioned rapid detection method in real samples, corn flour, millet flour, and rice flour were selected for recovery rate determination; the results are shown in Table 1. Table 1 shows the recovery rates of the assembled products after adding different concentrations of aflatoxin B1 to the pretreated food samples.

[0066] Table 1

[0067]

[0068] As shown in Table 1, the recoveries of aflatoxin B1 after adding different concentrations ranged from 94.61% to 109.92% (RSD < 5%), indicating that the method has good accuracy.

[0069] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A perylene imide derivative / sodium dodecyl sulfonate assembly, wherein the perylene imide derivative has the structure shown in formula (I), and the sodium dodecyl sulfonate has the structure shown in formula (II): Formula (I); Formula (II).

2. The method for preparing the perylene imide derivative / sodium dodecyl sulfonate assembly according to claim 1, characterized in that, Includes the following steps: S1) The compound shown in formula (III) is mixed with pyridine to carry out the first reaction, and the compound shown in formula (IV) is obtained; Formula (Ⅲ); Formula (Ⅳ); S2) The compound shown in formula (Ⅳ) and the compound shown in formula (Ⅴ) are reacted in the presence of imidazole to obtain the compound shown in formula (I); Formula (V); Equation (I); S3) The compound shown in formula (I) is mixed with the compound shown in formula (II) to carry out the third reaction, and a perylene imide derivative / sodium dodecyl sulfonate assembly is obtained; Formula (II).

3. The preparation method according to claim 2, characterized in that, The temperature of the first reaction is 70-80℃, and the reaction time is 4-6h.

4. The preparation method according to claim 2, characterized in that, The temperature of the second reaction is 90-120℃, and the reaction time is 8-10h.

5. The preparation method according to claim 2, characterized in that, After the second reaction is completed, the system undergoes post-processing: The resulting reaction mixture was subjected to solvent removal and precipitation treatment in sequence to obtain the compound shown in formula (I).

6. The preparation method according to claim 5, characterized in that, The precipitation process specifically includes: precipitating the product with hydrochloric acid, centrifuging, washing the precipitate with water, and then vacuum drying it.

7. The preparation method according to claim 2, characterized in that, The temperature of the third reaction is 25°C, and the reaction time is 30 minutes.

8. The use of the perylene imide derivative / sodium dodecyl sulfonate assembly according to claim 1 in the preparation of aflatoxin B1 detection reagents, test strips or kits.

9. A method for detecting aflatoxin B1 in food, characterized in that, The perylene imide derivative / sodium dodecyl sulfonate assembly of claim 1 was used as a probe.

10. A reagent for detecting aflatoxin B1, characterized in that, Includes the perylene imide derivative / sodium dodecyl sulfonate assembly as described in claim 1.

11. A test strip for detecting aflatoxin B1, characterized in that, Includes the perylene imide derivative / sodium dodecyl sulfonate assembly as described in claim 1.

12. A kit for detecting aflatoxin B1, characterized in that, Includes the perylene imide derivative / sodium dodecyl sulfonate assembly as described in claim 1.

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