Accurate and rapid aflatoxin B1 detection method based on double-excitation fluorescence response mode

Abstract

The invention provides a rapid aflatoxin B1 detection method based on a double-excitation-co-assembly fluorescent probe, and relates to the field of rapid detection of grain mycotoxins. The method comprises the following specific steps: preparing the perylenetetracarboxylic acid / ruthenium derivative co-assembled fluorescent probe, constructing a double-excitation fluorescence response platform, acquiring a fluorescence signal, constructing a quantitative model and detecting an actual sample. According to the invention, a fluorescent probe is combined with a double-signal fluorescent channel, and the detection sensitivity of aflatoxin B1 is greatly improved by utilizing different fluorescent responses of the probe to toxins under different excitation wavelengths; the double-excitation type fluorescent probe is used for rapid detection of aflatoxin B1 for the first time, and compared with other single-channel response methods, the double-excitation type fluorescent probe has the advantages of being high in accuracy, high in detection sensitivity and the like. The method can be applied to detection of aflatoxin B1 in food.

Description

Technical Field

[0001] The invention relates to the technical field of biosensor detection, and in particular to a fluorescence dual-excitation rapid detection method for aflatoxin B1 of a perylenetetracarboxylic acid / ruthenium derivative co-assembly. Background Art

[0002] Aflatoxin B1 is a secondary metabolite of Aspergillus flavus and Aspergillus parasiticus. It is the most widely distributed, most toxic and most harmful aflatoxin, and has strong thermal and chemical stability. The 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 food safety. Due to the weak interaction between aflatoxin B1 and the sensor probe, the development of signal amplification technology is crucial 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 professional technical laboratory researchers to conduct detection, which is time-consuming and, to a certain extent, limits the development of aflatoxin B1 detection using traditional methods.

[0004] The interaction between supramolecular recognition systems and small molecules is mainly non-covalent bond changes, which have the advantages of easy synthesis and good chemical stability, providing multiple opportunities for the development of new supramolecular chemical sensors for aflatoxin B1. However, due to the limitations of receptor binding affinity and complex environment, more clever assembly strategies and more precise signal amplification strategies need to be explored.

[0005] In order to overcome the shortcomings of existing detection methods, the present invention proposes to combine 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] The present invention aims at the problems existing in the existing detection technology and provides a fluorescence rapid detection method of aflatoxin B1 based on a dual excitation fluorescence response mode. The perylene tetracarboxylic acid / ruthenium derivative co-assembly provided by the present invention has good water stability, can produce different color responses to aflatoxin B1 under different excitation wavelengths, and can achieve high sensitivity and specificity detection of aflatoxin B1. The dual-channel aflatoxin B1 detection method provided by the present invention has the advantages of low detection limit, high detection efficiency, etc., is suitable for actual samples, and is easy to use.

[0007] In order to achieve the above object, the technical solution adopted by the present invention includes a rapid fluorescence detection method of aflatoxin B1 based on a dual excitation fluorescence response mode, comprising the following steps:

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

[0009] S1, mixing 3,4,9,10-pyrenetetracarboxylic acid with 1,4-dibromobutane / 1,6-dibromohexane / 1,8-dibromooctane, and performing a first reaction in the presence of hexadecyltrimethylammonium bromide;

[0010] S2, mixing the above product with trimethylamine, and performing a second reaction to obtain a compound represented by formula (I);

[0011]

[0012] S3, mixing the compound represented by formula (I) with terpyridine ruthenium chloride, and performing a third reaction to obtain a perylene tetracarboxylic acid / ruthenium derivative co-assembled fluorescent probe;

[0013] Step 2: prepare a standard solution of aflatoxin B1, and add different volumes of the standard solution of aflatoxin B1 to the dual excitation probe solution to obtain mixed solutions of different concentrations, wherein the pH of the solution is 7.8-8.2;

[0014] Step 3: S1. Under an excitation wavelength of 440 nm, the fluorescence spectra of the solutions of step 2 with different concentrations were measured using a fluorescence spectrometer, and the fluorescence intensities I at emission wavelengths of 490 nm and 605 nm were recorded. 490 and I 605 , calculate the fluorescence change I under the above excitation wavelength 605 / I 490 , denoted as F1;

[0015] S2. Under an excitation wavelength of 365 nm, the fluorescence spectra of the solutions of step 2 with 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 under the above excitation wavelength 440 / I 605 +I 490 / I 605 , recorded as F2;

[0016] S3. Combine the signals of the above two wavelengths, use the concentration of aflatoxin B1 as the horizontal coordinate and F1×F2 as the vertical coordinate to establish a detection standard curve;

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

[0018] Preferably, in the present invention, the reaction temperature in S1 of step 1 is 120-130° C., more preferably 120° C.; and the reaction time is 3-5 h, more preferably 3 h.

[0019] Preferably, in the present invention, the reaction temperature in S2 of step 1 is 80-90° C., more preferably 80° C.; the reaction time is 72-80 h, more preferably 72 h.

[0020] Preferably, after the S1 reaction in step 1 is completed, the system is post-treated:

[0021] The obtained reaction mixture is subjected to solvent removal treatment and precipitation treatment in sequence to obtain the compound represented by formula (I).

[0022] Preferably, the precipitation treatment comprises: precipitating the product with hydrochloric acid, washing the precipitated product with water after centrifugation and then vacuum drying.

[0023] Preferably, in the present invention, the molar ratio of the S3 perylene tetracarboxylic acid derivative to ruthenium pyridine in step 1 is 1:350.

[0024] Preferably, the pH of the solution in step 2 is 7.8-8.2, more preferably 8.0.

[0025] The reason why the perylenetetracarboxylic acid / pyridine ruthenium assembly provided by the present invention has excellent aflatoxin B1 detection performance may be based on the following principle: the perylenetetracarboxylic acid / pyridine ruthenium assembly of the present invention can be recognized by aflatoxin B1 through non-covalent forces such as electrostatic attraction, hydrophobic interaction, and hydrogen bonding, and the two form a stable supramolecular aggregate in aqueous solution, resulting in a change in the probe fluorescence signal.

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

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

[0028]

[0029] Compared with the existing technology, its advantages and positive effects are:

[0030] (1) The perylene tetracarboxylic acid derivative provided by the present invention is a small molecule cationic probe, comprising a perylene ring structure with good optical activity and a side chain capable of ensuring good solubility;

[0031] (2) The conformation and aggregation state of the perylenetetracarboxylic acid / pyridine ruthenium assembly provided by the present invention in the solution show different changes 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 perylenetetracarboxylic acid / ruthenium pyridine assembly has a 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%. It has the advantages of being fast, accurate, simple to operate, and easy to visualize. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The fluorescence changes of the perylene tetracarboxylic acid / pyridine ruthenium assembly probe after adding different concentrations of aflatoxin B1 into Tris-HCl buffer (0.1 mol / L, pH=8.0);

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

[0035] The present invention has no particular limitation on the source of the perylenetetracarboxylic acid / ruthenium pyridine assembly, which can be commercially available or prepared by methods well known to those skilled in the art.

[0036] To further illustrate the present invention, the fluorescence rapid detection method of aflatoxin B1 based on perylenetetracarboxylic acid / pyridineruthenium assembly provided by the present invention is described in detail below in conjunction with examples.

[0037] Preparation of spare materials:

[0038] Preparation of buffer solution: Weigh tris(hydroxymethyl)aminomethane (Tris) solid, prepare 100 mL of 0.1 mol / L Tris solution with distilled water, adjust its pH to 8.0 with 0.1 mol / L hydrochloric acid standard solution, and store it in a refrigerator at 4°C for later use.

[0039] Preparation of probe mother solution: Perylene tetracarboxylic acid and ruthenium pyridine represented by formula (I) and formula (II) were prepared into mother solutions with concentrations of 0.1mmol / L and 1mmol / L respectively with distilled water, and the solutions were divided into solutions of the same volume in small bottles for later use. Before the spectral test, the mother solutions were mixed for the third reaction, and the prepared Tris-HCl buffer solution (0.1mol / L, pH=8.0) was used to dilute to a certain concentration for the test.

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

[0041] Preparation of actual samples: Cereal samples were purchased from a local supermarket, extracted by ultrasound, centrifuged, and filtered through a 0.22 μm microporous membrane, and the filtrate was used in the experiment.

[0042] Example 1

[0043] Preparation of the compound represented by formula (I):

[0044] Dissolve 3,4,9,10 perylene tetracarboxylic acid (1 mmol) in 30 mL of water, add hexadecyl trimethyl ammonium bromide (CTAB, 400 mg, 1.1 mmol), stir magnetically for 10 min, then add excess 1,6-dibromohexane [Br(CH2)6Br, 4.3 g, 20.0 mmol], and stir vigorously at 125°C for 3 h. After the reaction is completed, let stand until the liquid in the flask is clearly separated, add 30 mL of chloroform to the flask, separate the aqueous phase and the organic phase with a separatory funnel, retain the organic phase and wash it repeatedly with NaCl solution (15%) 3 times. Remove the solvent from the washed solution by rotary evaporation to obtain intermediate 1;

[0045] 484 mg of intermediate product 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 several times and the reaction progress was continuously monitored by thin layer chromatography. After the reaction was closed, the reaction solution was allowed to stand and cool to room temperature, and the solvent was removed by rotary evaporation and dried in a vacuum oven to obtain an orange-red solid final product.

[0046] The compound represented by formula (I) is well soluble in water. The compound represented by formula (I) is ultrasonically reacted with ruthenium pyridinium trichloride for 30 minutes at 25° C. (molar concentration ratio is 1:350) to obtain a perylenetetracarboxylic acid / ruthenium pyridinium assembly (abbreviated as probe or assembly).

[0047] Example 2

[0048] Fluorescence spectrum test:

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

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

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

[0052] The aforementioned assembly is excited at an excitation wavelength of 440nm, emitting a maximum emission peak at 490nm. With the addition of aflatoxin B1, the fluorescence emission intensity of the aforementioned assembly gradually decreases, and the fluorescence color changes from green to red. The aforementioned assembly is excited at an excitation wavelength of 365nm, emitting a maximum emission peak at 605nm. With the addition of aflatoxin B1, the fluorescence emission intensity of the aforementioned assembly gradually increases at 440nm and 490nm, and the fluorescence color changes from red to blue. According to the detection limit calculation method, the detection limit of the assembly for aflatoxin B1 is 0.12ng / mL.

[0053] Example 3

[0054] Optional Studies:

[0055] Select possible interferences in food samples containing aflatoxin B1, including common mycotoxins, anions, cations, sugars and amino acids: K + , Ca 2+ 、Na + Mg 2+ 、Al 3+ 、Zn 2+ , Fe 3+, glucose, lactose, aspartic acid, tryptophan, phenylalanine, alanine, histidine, lysine, vomitoxin, zearalenone, fumonisin B1. In the test, the concentration of the perylene tetracarboxylic acid derivative was 0.1 μmol / L, the concentration of ruthenium pyridine was 35 μmol / L, and the concentration of all interferents was 5 μg / mL. The fluorescence spectrum was tested under the same test conditions. The fluorescence intensity of the assembly at 490 nm and 605 nm before and after the addition of the interferent was taken as the ratio, I 490 / I 605 As a parameter to measure the influence of the aforementioned assembly on the analyte.

[0056] The results are as follows Figure 2 As shown (that is, Figure 2 Figure 3 shows the relative absorption values ​​of the above-mentioned assembly interacting with aflatoxin B1 and other interfering substances in Tris-HCl (0.1 mol / L, pH=8.0) buffer.

[0057] It can be seen from the figure that except for aflatoxin B1, the I 605 / I 490 ≈0.3, which is much lower than aflatoxin B1. This result shows that the aforementioned assembly has excellent selectivity for aflatoxin B1.

[0058] Example 4

[0059] Application of actual samples: In order to verify the feasibility of the above method in actual samples, rice flour, corn flour and wheat flour were selected for the determination of recovery rate; the results are shown in Table 1. Table 1 shows the recovery rate after adding different concentrations of aflatoxin B1 to the pre-treated food samples.

[0060] Table 1

[0061] sample Spike concentration (μg / mL) Detection value (μg / mL) Recovery rate (%) Standard deviation (%) 5 4.99 99.9 3.4 Rice flour 10 9.32 93.2 4.5 20 18.94 94.7 3.9 5 531 106.2 4.2 Corn flour 10 9.21 92.1 2.5 20 21.13 105.6 4.9 5 4.4 90.1 5.7 wheat flour 10 9.5 95.1 4.3 20 18.8 94.2 3.9

[0062] As shown in Table 1, after adding different concentrations of aflatoxin B1, the spiked recoveries were between 90.1% and 106.2% (RSD < 5%), indicating that the method had good accuracy.

[0063] The above embodiments are only used to help understand the method and core idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A rapid detection method for aflatoxin B1 based on dual excitation-co-assembled fluorescent probes, characterized in that: The following steps are involved: Step 1: preparing a perylenetetracarboxylic acid / ruthenium derivative co-assembly as a fluorescent probe; Step 2: prepare a standard solution of aflatoxin B1, and add different volumes of the standard solution of aflatoxin B1 to the dual excitation probe solution to obtain mixed solutions of different concentrations, wherein the pH of the solution is 7.8-8.2; Step 3: S1. Under an excitation wavelength of 440 nm, the fluorescence spectra of the solutions of step 2 with different concentrations were measured using a fluorescence spectrometer, and the fluorescence intensities I at emission wavelengths of 490 nm and 605 nm were recorded. 490 and I 605 , calculate the fluorescence change I under the above excitation wavelength 605 / I 490 , denoted as F1; S2. Under an excitation wavelength of 365 nm, the fluorescence spectra of the solutions of step 2 with 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 under the above excitation wavelength 440 / I 605 +I 490 / I 605 , recorded as F2; S3. Combine the signals of the above two wavelengths, use the concentration of aflatoxin B1 as the horizontal coordinate and F1×F2 as the vertical coordinate to establish a detection standard curve; Step 4: Pretreat the sample to obtain a test sample solution, measure the fluorescence change intensity of the test sample solution, calculate the content of aflatoxin B1 in the test sample through the standard curve, and realize the detection of aflatoxin B1 in unknown samples.

2. According to claim 1, a rapid detection method of aflatoxin B1 based on dual excitation-co-assembled fluorescent probes, characterized in that: In step 1, the molar concentration ratio of the perylene tetracarboxylic acid derivative to ruthenium pyridine is 1:

350.

3. According to claim 1, a rapid detection method of aflatoxin B1 based on dual excitation-co-assembled fluorescent probes, characterized in that: The concentration range of aflatoxin B1 in step 3 S1 and S2 is 0-35 μg / mL.

4. According to claim 1, a rapid detection method of aflatoxin B1 based on dual excitation-co-assembled fluorescent probes, characterized in that: The fluorescence signal processing formula in step 3 S3 is: F=F1×F2 Where F1 and F2 are the fluorescence changes at excitation wavelengths of 440nm and 365nm respectively.

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

6. A reagent, test paper or kit for detecting aflatoxin B1, characterized in that: Perylenetetracarboxylic acid derivative / ruthenium pyridine co-assembly was used as a fluorescent probe.

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