A precise and rapid detection method for aflatoxin B1 based on a dual-excitation fluorescence response mode

Abstract

This invention provides a rapid detection method for aflatoxin B1 based on a dual-excitation co-assembled fluorescent probe, relating to the field of rapid detection of mycotoxins in grains. The specific steps include: preparation of a perylenetetracarboxylic acid / ruthenium derivative co-assembled fluorescent probe, construction of a dual-excitation fluorescence response platform, acquisition of fluorescence signals, construction of a quantitative model, and detection of actual samples. This invention combines a fluorescent probe with a dual-signal fluorescence channel, utilizing the probe's different fluorescence responses to the toxin at different excitation wavelengths, significantly improving the detection sensitivity of aflatoxin B1. This invention is the first to use a dual-excitation fluorescent probe for rapid detection of aflatoxin B1, offering advantages such as high accuracy and high detection sensitivity compared to other single-channel response methods. It can be applied to the detection of aflatoxin B1 in food.

Description

Technical Field

[0001] This invention relates to the field of biosensing and detection technology, specifically to a rapid fluorescence dual-excitation detection method for aflatoxin B1 in a perylenetetracarboxylic acid / ruthenium derivative co-assembly. Background Technology

[0002] Aflatoxin B1, a secondary metabolite of Aspergillus flavus and Aspergillus parasiticus, is the most widely distributed, most toxic, and most harmful aflatoxin, exhibiting strong thermal and chemical stability. Rapid and accurate detection of aflatoxin B1 in food samples, such as wheat, is crucial for the prevention of persistent organic contaminants and for ensuring food safety. Due to the weak interaction between aflatoxin B1 and the sensing probe, the development of signal amplification techniques is essential for the rapid and accurate detection of aflatoxin B1.

[0003] Traditional detection methods (liquid chromatography, enzyme-linked immunosorbent assay, etc.) rely on expensive instruments and specialized technical laboratory researchers, and the detection time is relatively long, which to some extent limits the development of aflatoxin B1 detection using traditional methods.

[0004] The interaction between supramolecular recognition systems and small molecules is mainly based on non-covalent bond changes, offering advantages such as easy synthesis and good chemical stability, providing various opportunities for the development of novel aflatoxin B1 supramolecular chemical sensors. However, limitations imposed by receptor binding affinity and complex environments necessitate the exploration of more ingenious assembly strategies and more precise signal amplification strategies.

[0005] To overcome the shortcomings of existing detection methods, this invention proposes a combination of supramolecular co-assembly strategy and dual-excitation fluorescence sensing signal amplification technology for the accurate detection of aflatoxin B1 in food samples. Summary of the Invention

[0006] This invention addresses the problems of existing detection technologies by providing a rapid fluorescence detection method for aflatoxin B1 based on a dual-excitation fluorescence response mode. The perylenetetracarboxylic acid / ruthenium derivative co-assembly provided by this invention exhibits good water stability and can produce different color responses to aflatoxin B1 at different excitation wavelengths, enabling highly sensitive and specific detection of aflatoxin B1. The dual-channel aflatoxin B1 detection method provided by this invention has advantages such as low detection limit and high detection efficiency, is suitable for practical samples, and is easy to apply.

[0007] To achieve the above objectives, the technical solution adopted by the present invention includes a rapid fluorescence detection method for aflatoxin B1 based on a dual-excitation fluorescence response mode, comprising the following steps:

[0008] Step 1: Preparation of perylenetetracarboxylic acid / ruthenium derivative assemblies;

[0009] S1. 3,4,9,10-pyrenetetracarboxylic acid is mixed with 1,4-dibromobutane / 1,6-dibromohexane / 1,8-dibromooctane, and the first reaction is carried out in the presence of hexadecyltrimethylammonium bromide;

[0010] S2. The above product is mixed with trimethylamine to carry out a second reaction, yielding the compound shown in formula (I);

[0011] Formula (I);

[0012] S3. The compound shown in formula (I) is mixed with terpyridine ruthenium chloride and subjected to the third reaction to obtain a perylene tetracarboxylic acid / ruthenium derivative co-assembled fluorescent probe;

[0013] Step 2: Prepare a standard solution of aflatoxin B1. Add different volumes of the aflatoxin B1 standard solution to the dual-excitation probe solution to obtain mixed solutions of different concentrations. The pH of the solution is 7.8-8.2.

[0014] Step 3: S1. At an excitation wavelength of 440 nm, measure the fluorescence spectra of the solutions described in Step 2 at different concentrations using a fluorescence spectrometer, and record the fluorescence intensity I at emission wavelengths of 490 nm and 605 nm. 490 and I 605 Calculate the fluorescence change I at the above excitation wavelength. 605 / I 490 , denoted as F1;

[0015] S2. At an excitation wavelength of 365 nm, the fluorescence spectra of the solutions described in step two at different concentrations were measured using a fluorescence spectrometer, and the fluorescence intensities I at emission wavelengths of 440 nm, 490 nm, and 605 nm were recorded. 440 I 490 and I 605 Calculate the fluorescence change I at the above excitation wavelength. 440 / I 605 +I 490 / I 605 , denoted as F2;

[0016] S3. Combining the signals from the two wavelengths mentioned above, a standard curve for detection is established with the concentration of aflatoxin B1 as the x-axis and F1×F2 as the y-axis.

[0017] Step 4: Pre-treat the sample to obtain the test sample solution, measure the fluorescence change intensity of the test sample solution, and calculate the content of aflatoxin B1 in the test sample through the standard curve to realize the detection of aflatoxin B1 in unknown samples.

[0018] Preferably, in step S1 of this invention, the reaction temperature is 120-130℃, more preferably 120℃; and the reaction time is 3-5 h, more preferably 3 h.

[0019] Preferably, in step S2 of the present invention, the reaction temperature is 80-90℃, more preferably 80℃; and the reaction time is 72-80 h, more preferably 72 h.

[0020] Preferably, after the S1 reaction in step one of this invention is completed, the system undergoes post-processing:

[0021] The resulting reaction mixture was subjected to solvent removal and precipitation treatment in sequence to obtain the compound shown in formula (I).

[0022] 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.

[0023] Preferably, in this invention, the molar ratio of the S3 perylenetetracarboxylic acid derivative to ruthenium pyridine in step one is 1:350.

[0024] Preferably, the pH of the solution in step two is 7.8-8.2, more preferably 8.0.

[0025] The excellent aflatoxin B1 detection performance of the perylenetetracarboxylic acid / pyridineruthenium assembly provided by this invention may be based on the following principle: the perylenetetracarboxylic acid / pyridineruthenium assembly of this invention can recognize aflatoxin B1 through non-covalent interactions such as electrostatic attraction, hydrophobic interaction, and hydrogen bonding. In aqueous solution, the two form a stable supramolecular aggregate, which leads to a change in the probe fluorescence signal.

[0026] Experimental results show that the detection method provided by this invention has high detection efficiency and sensitivity.

[0027] The present invention also provides a reagent, test strip or kit for detecting aflatoxin B1, comprising a perylenetetracarboxylic acid / pyridineruthenium assembly of formula (I) and terpyridineruthenium chloride;

[0028] Formula (I).

[0029] Compared with existing technologies, its advantages and positive effects are as follows:

[0030] (1) The perylene tetracarboxylic acid derivative 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;

[0031] (2) The perylenetetracarboxylic acid / pyridineruthenium assembly provided by the present invention exhibits different changes in conformation and aggregation state in solution in response to external stimuli, thereby causing changes in its optical properties, and has a dual-excitation fluorescence sensing mode with signal superposition and self-calibration.

[0032] (3) The above perylenetetracarboxylic acid / pyridineruthenium assembly has specific recognition ability for aflatoxin B1, with a detection limit as low as 0.12 ng / mL. The spiked recovery rate of the grain sample is 90-110%, which has the advantages of being fast, accurate, simple to operate and easy to visualize. Attached Figure Description

[0033] Figure 1 Fluorescence changes of perylenetetracarboxylic acid / ruthenium pyridine assembly probes after adding different concentrations of aflatoxin B1 to Tris-HCl buffer (0.1 mol / L, pH=8.0);

[0034] Figure 2 The relative fluorescence intensity of the perylenetetracarboxylic acid / pyridineruthenium assembly in Tris-HCl buffer (0.1 mol / L, pH=8.0) interacting with different substances is shown. Detailed Implementation

[0035] The present invention does not have any particular limitation on the source of the above perylenetetracarboxylic acid / pyridineruthenium assembly, which can be commercially available or prepared according to methods known to those skilled in the art.

[0036] To further illustrate the present invention, the rapid fluorescence detection method for aflatoxin B1 based on the perylenetetracarboxylic acid / pyridineruthenium assembly provided by the present invention will be described in detail below with reference to embodiments.

[0037] Preparation of spare materials:

[0038] Preparation of buffer solution: Weigh tris(hydroxymethyl)aminomethane (Tris) solid and prepare 100 mL of 0.1 mol / L Tris solution with distilled water. Adjust the pH to 8.0 with 0.1 mol / L hydrochloric acid standard solution. Store at 4°C for later use.

[0039] Preparation of probe stock solutions: Perylenetetracarboxylic acid and ruthenium pyridine, as shown in formulas (I) and (II), were prepared into stock solutions with concentrations of 0.1 mmol / L and 1 mmol / L, respectively, using distilled water. These solutions were then dispensed into equal volumes into vials for later use. Before spectral testing, the stock solutions were mixed and subjected to the third reaction. During spectral testing, the solutions were diluted to a specific concentration using a prepared Tris-HCl buffer solution (0.1 mol / L, pH=8.0) for testing.

[0040] Preparation of analytes and interfering substances: Aflatoxin B1 was prepared into a 1 mg / mL stock solution using dimethyl sulfoxide. Other interfering substances were prepared into 1 mg / mL stock solutions using distilled water. These solutions were stored at 4°C for later use.

[0041] Preparation of actual samples: Grain samples were purchased from a local supermarket, extracted by ultrasonication, centrifuged, filtered through a 0.22 μm microporous membrane, and the filtrate was used for experiments.

[0042] Example 1

[0043] Preparation of the compound shown in formula (I):

[0044] 3,4,9,10-perylenetetracarboxylic acid (1 mmol) was dissolved in 30 mL of water, and hexadecyltrimethylammonium bromide (CTAB, 400 mg, 1.1 mmol) was added. The mixture was magnetically stirred for 10 min, and then excess 1,6-dibromohexane [Br(CH2)6Br, 4.3 g, 20.0 mmol] was added. The mixture was stirred vigorously under reflux at 125 °C for 3 h. After the reaction was completed, the mixture was allowed to stand until the liquid in the flask clearly separated into layers. 30 mL of chloroform was added to the flask, and the aqueous and organic phases were separated using a separatory funnel. The organic phase was retained and washed three times repeatedly with NaCl solution (15%). The solvent was removed from the washed solution by rotary evaporation to obtain intermediate product 1.

[0045] 484 mg of intermediate 1 (0.5 mmol), 50 mL of tetrahydrofuran, and 5 mL of trimethylamine aqueous solution (30%) were placed in a 100 mL flask and refluxed at 80 °C for 3 days. During the reaction, purified water (15 mL in total) was added in several portions, and the reaction progress was continuously monitored by thin-layer chromatography. After the reaction was stopped, the reaction solution was allowed to cool to room temperature, the solvent was removed by rotary evaporation, and then dried in a vacuum oven to obtain an orange-red solid final product.

[0046] The compound shown in formula (I) is well soluble in water. The compound shown in formula (I) was reacted with ruthenium trichloride pyridine by sonication at 25°C for 30 min (molar concentration ratio of 1:350) to obtain a perylenetetracarboxylic acid / ruthenium pyridine assembly (referred to as probe or assembly).

[0047] Example 2

[0048] Fluorescence spectroscopy testing:

[0049] Take 90 μL of probe stock solution and 910 μL of Tris-HCl buffer (0.1 mol / L) and add them to a 1 mL sample cell. After mixing thoroughly, measure the fluorescence spectrum of the probe buffer and record the change in fluorescence intensity at an excitation wavelength of 440 nm as F1(I). 605 / I490 The fluorescence intensity change at an excitation wavelength of 365 nm is F2 (I 440 / I 605 +I 490 / I 605 Subsequently, aflatoxin B1 of a certain concentration gradient was gradually added to the sample cell, and after mixing evenly, the corresponding fluorescence spectrum was measured. The signals of the two wavelengths were combined, and a detection standard curve was established with the concentration of aflatoxin B1 as the abscissa and F1×F2 as the ordinate.

[0050] The concentration range of the aflatoxin B1 standard solution is 0-30 μg / mL.

[0051] The results are as follows Figure 1 As shown (that is, Figure 1 This indicates the variation of fluorescence intensity of the aforementioned assembly at different excitation wavelengths with aflatoxin B1 concentration in Tris-HCl (0.1 mol / L, pH=8.0) buffer. The slit width for both excitation and emission light was 3.0 nm.

[0052] The aforementioned assembly, when excited at a wavelength of 440 nm, emitted a maximum emission peak at 490 nm. With the addition of aflatoxin B1, the fluorescence emission intensity of the assembly gradually decreased, exhibiting a fluorescence color change from green to red. When excited at a wavelength of 365 nm, the assembly emitted a maximum emission peak at 605 nm. With the addition of aflatoxin B1, the fluorescence emission intensity of the assembly gradually increased at 440 nm and 490 nm, exhibiting a fluorescence color change from red to blue. Based on the method for calculating the detection limit, the detection limit of the assembly for aflatoxin B1 is 0.12 ng / mL.

[0053] Example 3

[0054] Selective studies:

[0055] Potential interfering substances in food samples containing aflatoxin B1 were selected, including common mycotoxins, anions, cations, sugars, and amino acids: K. + Ca 2+ Na + Mg 2+ Al 3+ Zn 2+ Fe 3+The substances tested included glucose, lactose, aspartic acid, tryptophan, phenylalanine, alanine, histidine, lysine, vomitoxin, zearalenone, and fumonisin B1. The concentrations of the perylenetetracarboxylic acid derivatives were 0.1 μmol / L, ruthenium pyridine was 35 μmol / L, and all interfering substances were 5 μg / mL. Fluorescence spectra were measured under the same conditions. The ratio of the fluorescence intensities of the assemblies before and after the addition of the interfering substances at 490 nm and 605 nm was calculated. 605 / I 490 This serves as a parameter to measure the degree of influence of the aforementioned assembly on the test object.

[0056] 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 substances in Tris-HCl (0.1 mol / L, pH=8.0) buffer.

[0057] As can be seen from the figure, except for aflatoxin B1, the I of all other substances... 605 / I 490 The concentration of aflatoxin B1 was approximately 0.3, which is much lower than that of aflatoxin B1. This result indicates that the aforementioned assembly has excellent selectivity for aflatoxin B1.

[0058] Example 4

[0059] Application to real samples: To verify the feasibility of the aforementioned method in real samples, rice flour, corn flour, and wheat flour were selected for recovery rate determination; the results are shown in Table 1. Table 1 shows the recovery rates of aflatoxin B1 after adding different concentrations to the pretreated food samples.

[0060] Table 1

[0061]

[0062] As shown in Table 1, the spiked recoveries of aflatoxin B1 at different concentrations ranged from 90.1% to 106.2% (RSD < 5%), indicating that the method has good accuracy.

[0063] 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 rapid detection method for aflatoxin B1 based on a dual-excitation-co-assembly fluorescent probe, characterized in that, Includes the following steps: Step 1: Prepare a perylenetetracarboxylic acid / ruthenium derivative co-assembly as a fluorescent probe: S1. 3,4,9,10-pyrenetetracarboxylic acid is mixed with 1,4-dibromobutane / 1,6-dibromohexane / 1,8-dibromooctane, and the first reaction is carried out in the presence of hexadecyltrimethylammonium bromide; S2. The above product is mixed with trimethylamine to carry out a second reaction, yielding the compound shown in formula (I); Formula (I); S3. The compound shown in formula (I) is mixed with terpyridine ruthenium chloride and subjected to the third reaction to obtain a perylene tetracarboxylic acid / ruthenium derivative co-assembled fluorescent probe; Step 2: Prepare a standard solution of aflatoxin B1. Add different volumes of the aflatoxin B1 standard solution to the dual-excitation probe solution to obtain mixed solutions of different concentrations. The pH of the solution is 7.8-8.

2. Step 3: S1. At an excitation wavelength of 440 nm, measure the fluorescence spectra of the solutions described in Step 2 at different concentrations using a fluorescence spectrometer, and record the fluorescence intensity I at emission wavelengths of 490 nm and 605 nm. 490 and I 605 Calculate the fluorescence change I at the above excitation wavelength. 605 / I 490 , denoted as F1; S2. At an excitation wavelength of 365 nm, the fluorescence spectra of the solutions described in step two at different concentrations were measured using a fluorescence spectrometer, and the fluorescence intensities I at emission wavelengths of 440 nm, 490 nm, and 605 nm were recorded. 440 I 490 and I 605 Calculate the fluorescence change I at the above excitation wavelength. 440 / I 605 +I 490 / I 605 , denoted as F2; S3. Combining the signals from the two wavelengths mentioned above, a standard curve for detection is established with the concentration of aflatoxin B1 as the x-axis and F1×F2 as the y-axis. Step 4: Pre-treat the sample to obtain the test sample solution, measure the fluorescence change intensity of the test sample solution, and calculate the content of aflatoxin B1 in the test sample through the standard curve to realize the detection of aflatoxin B1 in unknown samples.

2. The rapid detection method for aflatoxin B1 based on dual-excitation-co-assembly fluorescent probes according to claim 1, characterized in that, In step one, the molar ratio of the compound shown in formula (I) to ruthenium terpyridine chloride is 1:

350.

3. The rapid detection method for aflatoxin B1 based on dual-excitation-co-assembly fluorescent probes according to claim 1, characterized in that, In step three, the concentration range of aflatoxin B1 in S1 and S2 is 0-35 μg / mL.

4. The rapid detection method for aflatoxin B1 based on dual-excitation-co-assembly fluorescent probes according to claim 1, characterized in that, In step S3, when establishing the detection standard curve, the fluorescence signal processing formula is as follows: F = F1 × F2 F1 and F2 represent the fluorescence changes at excitation wavelengths of 440 nm and 365 nm, respectively.

5. The rapid detection method for aflatoxin B1 based on dual-excitation-co-assembly fluorescent probes according to claim 1, characterized in that, The fluorescent probe changes its fluorescence color from green to red when the excitation wavelength is 440 nm, and changes its fluorescence color from red to blue when the excitation wavelength is 365 nm.

6. A reagent, test strip, or kit for detecting aflatoxin B1, characterized in that, Perylenetetracarboxylic acid derivative / pyridineruthenium co-assembly was used as a dual-excitation-co-assembly fluorescent probe; The perylene tetracarboxylic acid derivative / pyridine ruthenium co-assembly is the perylene tetracarboxylic acid / ruthenium derivative co-assembled fluorescent probe as described in any one of claims 1 to 5.

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