Azobisformamide ratio fluorescence detection method based on CoOOH nanosheet loaded Au nano-cluster material

Abstract

The invention discloses an azodicarbonamide ratio fluorescence detection method based on a CoOOH nanosheet loaded Au nano-cluster material, and belongs to the technical field of fluorescence sensing. The CoOOH nanosheet loaded Au nano-cluster material provided by the invention is prepared by carrying out a one-step electrostatic assembly method on AuNCs and a CoOOH nanosheet. The CoOOH nanosheet loaded Au nano-cluster material has the fluorescence property of the AuNCs and the biomimetic catalytic performance of the CoOOH nanosheet at the same time. The invention further provides an azodicarbonamide ratio fluorescence detection method based on the CoOOH nanosheet loaded Au nano-cluster material, and compared with an existing detection method, the azodicarbonamide ratio fluorescence detection method has the remarkable advantages that the method is high in selectivity; by utilizing a ratio fluorescence mechanism, interference caused by environment and instrument fluctuation can be effectively reduced, reliable quantitative analysis of azodicarbonamide is realized, and the method has the advantages of high sensitivity and high credibility and has a wide prospect in the field of fluorescence sensing detection.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescence sensing, and in particular relates to an azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Azodicarbonamide (ADC) is widely considered a food additive and is present in products used as a flour bleaching agent or gluten fortifier. ADC can decompose into trace amounts of diphenylcarbamide and semicarbazide during heating, which greatly increase the risk of carcinogenicity, teratogenicity and mutagenicity. Therefore, accurate and sensitive detection of ADC content is essential to ensure the quality of flour products.

[0004] For the detection of azodicarbonamide (azoformamide) in flour, commonly used methods include high performance liquid chromatography (HPLC), fluorescence colorimetry, spectrophotometry, infrared spectroscopy, hyperspectral imaging, terahertz time-domain spectroscopy, Raman spectroscopy, etc. (Tian Linshuang et al., "Research Progress on Detection Methods of Azodicarbonamide in Wheat Flour and Its Products"). Traditional chemical detection methods have high detection accuracy and low detection limit. Some methods have a long inspection process, cumbersome steps and damage to samples. They can only be used in random inspections by quality inspection departments and cannot be used to detect all agricultural products. Among them, fluorescence analysis technology has attracted attention due to its simple and easy operation and cost-effectiveness. The currently proposed fluorescence method relies on the change of a single fluorescence signal and may be easily interfered by uncontrollable factors such as probe concentration, photobleaching and changes in environmental conditions. In order to overcome the problems faced by the single-emission fluorescence method, ratio fluorescence is the most effective strategy. Ratio fluorescence can provide self-calibration function and high selectivity, so it performs well in sensing detection.

[0005] CoOOH nanosheets, as two-dimensional hexagonal transition metal oxyhydroxide nanomaterials, show great potential in molecular detection. The main advantage of CoOOH nanosheets over other 2D nanomaterials is that they can be prepared in a short time under mild conditions without the need for expensive instruments. CoOOH nanosheets can act as an effective quencher to quench fluorescent molecules adsorbed on their surface. Chu et al. first developed a CoOOH-modified upconversion nanosystem, which utilized the efficient fluorescence quenching ability of CoOOH nanosheets and their ability to be reduced by ascorbic acid (AA) for the fluorescence detection of AA activity in human serum; CoOOH nanosheets also have inherent peroxidase-like activity and have been used to detect glucose, uric acid, cholesterol, etc.

[0006] Hu et al. prepared gold nanoclusters (AuNCs) with low peroxidase-like activity by using glutathione (GSH) as a reducing agent and stabilizer. Similar to the principle of aggregation-induced fluorescence enhancement, the aggregation of AuNCs promoted the interaction between internal complexes (e.g., van der Waals forces and lipophilic interactions) and increased the restrictions on the intramolecular vibration and rotation of the complex, thereby enhancing the catalytic activity of AuNCs, thereby achieving the degradation of Pb 2+ Analytical testing.

[0007] The patent publication "A fluorescent immunoassay method for imidacloprid based on gold nanoclusters anchored with cobalt oxyhydroxide nanosheets" (publication number: CN109580939A) discloses the use of glutathione-functionalized gold nanoclusters AuNCs and cobalt oxyhydroxide nanosheets (CoOOH NPs) to prepare CoOOH-AuNCs composite materials, and then detect imidacloprid. However, it is not possible to predict whether this technology is feasible for the detection of azodicarbonamide in flour. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides a ratio fluorescence detection method for azodicarbonamide based on CoOOH nanosheets loaded with Au nanocluster materials. The present invention uses AuNCs@CoOOH with fluorescent luminescence properties and enzyme-mimicking catalytic properties. The ratio fluorescence sensing method developed based on AuNCs@CoOOH can detect azodicarbonamide. The detection method has high sensitivity, high selectivity and simple preparation.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides an azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheet-loaded Au nanocluster material, wherein the CoOOH nanosheet-loaded Au nanocluster material comprises CoOOH nanosheets and Au nanoclusters loaded on the CoOOH nanosheets; the detection method comprises the following steps:

[0011] Step 1, preparing aqueous solutions containing different amounts of azodicarbonamide as standard solutions;

[0012] Step 2, adding the CoOOH nanosheet loaded with Au nanocluster material into a mixed solution containing the azodicarbonamide standard solution prepared in step 1, and a glutathione aqueous solution and a thiamine aqueous solution, and performing fluorescence detection after the reaction.

[0013] Preferably, with 365±5 nm as the excitation wavelength, the fluorescence emission intensities at 605±5 nm and 438±5 nm are collected, and the relationship between the ratio of the fluorescence intensities at 605±5 nm to 438±5 nm and the concentration of azodicarbonamide is established, thereby detecting the concentration of azodicarbonamide.

[0014] Preferably, the particle size of the CoOOH nanosheet-loaded Au nanocluster material is 50-80 nm.

[0015] The preparation method of the CoOOH nanosheet-loaded Au nanocluster material is to mix and stir an AuNCs aqueous solution with a CoOOH nanosheet aqueous solution, and obtain the material through a one-step electrostatic assembly method.

[0016] Preferably, in the preparation method, the mass concentration of the AuNCs aqueous solution is 0.3-0.5 mg / mL, and the mass concentration of the CoOOH nanosheets aqueous solution is 0.1-0.3 mg / mL.

[0017] Preferably, in the preparation method, the stirring reaction temperature is 20-35° C. and the reaction time is 25-40 min.

[0018] Preferably, in step 2 of the detection method, the mass concentration of the CoOOH nanosheet-loaded Au nanocluster material is 0.1-0.2 mg / mL, the concentration of the glutathione aqueous solution is 50-75 μM, and the concentration of the thiamine aqueous solution is 400-600 μM.

[0019] Preferably, in step 2 of the detection method, the reaction temperature is 45-65° C. and the reaction time is 25-45 min.

[0020] Preferably, the concentration range of azodicarbonamide detectable by the detection method is 0.1 to 50 μM.

[0021] Experiments show that the ratio fluorescence detection method provided by the present invention has the advantages of a wide linear range (0.1-50 μM), a detection limit as low as 0.034 μM (3.95 ppb), and simple operation for the detection of azodicarbonamide.

[0022] In a second aspect, the present invention also provides an application of an azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials in flour detection. In step 2, the azodicarbonamide concentration is used as the horizontal coordinate and the fluorescence intensity ratio F is used as the horizontal coordinate. 605 / F 438A standard curve is drawn as the ordinate; CoOOH nanosheets loaded with Au nanocluster materials are prepared, and added to flour aqueous solutions containing different spiked concentrations, and then glutathione aqueous solution and thiamine aqueous solution are added to the above mixed solution, and the reaction is carried out at 60°C for 40 minutes; 365nm is used as the excitation wavelength, the fluorescence intensity at 605nm and 438nm is recorded, and the fluorescence intensity ratio is calculated, and the ratio is substituted into the standard curve to calculate the content of azodicarbonamide in the actual sample to be tested.

[0023] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0024] (1) The present invention provides an azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials, which utilizes the CoOOH nanosheets loaded with Au nanocluster materials to have both the fluorescence properties of AuNCs and the enzyme-like catalytic properties of CoOOH nanosheets; the CoOOH nanosheets quench the fluorescence of AuNCs through fluorescence resonance energy transfer (FRET); in addition, the CoOOH nanosheets have oxidase-like properties and can catalyze the oxidation of non-fluorescent thiamine into fluorescent thiochromes.

[0025] (2) Compared with the existing detection methods of azodicarbonamide in flour, the ratio fluorescence detection method of azodicarbonamide based on CoOOH nanosheets loaded with Au nanocluster materials provided by the present invention has the following significant advantages: the method has high selectivity; the ratio fluorescence mechanism is used to effectively reduce the interference caused by environmental and instrument fluctuations, and realize reliable quantitative analysis of azodicarbonamide, which has the advantages of high sensitivity and high credibility, and has broad prospects in the field of fluorescence sensing detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 Transmission electron microscopy images of the CoOOH nanosheet-loaded Au nanocluster material prepared in Example 1 at different scales (where A is 100 nm and B is 10 nm), a scanning transmission electron microscopy image (C), and Co, Au, C, N, O and S element mapping images (DI).

[0028] Figure 2 The fluorescence emission spectra of AuNCs and AuNCs@CoOOH in Example 2 (A), the UV absorption graph and visible light photograph of AuNCs@CoOOH before and after catalysis of the chromogenic substrate (B), and the fluorescence emission image of AuNCs@CoOOH before and after catalysis of non-fluorescent thiamine (C).

[0029] Figure 3 The fluorescence emission spectra (A) and fluorescence intensity ratios (F) of the detection system after adding different concentrations of ADC solutions in Example 3 are shown in FIG. 605 / F 438 ) and the linear curve fitted with ADC concentration (B).

[0030] Figure 4 This is the selectivity of the ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials for azodicarbonamide in Example 4.

[0031] Figure 5 This is a schematic diagram of the mechanism of azodicarbonamide detection in the synthesized CoOOH nanosheet-loaded Au nanocluster material according to Example 1 of the present invention. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0034] The present invention provides a CoOOH nanosheet-loaded Au nanocluster material, and an azodicarbonamide ratio fluorescence detection method based on the CoOOH nanosheet-loaded Au nanocluster material, such as Figure 5 As shown in the figure, the CoOOH nanosheet-loaded Au nanocluster material is obtained by a one-step electrostatic assembly method of AuNCs and CoOOH nanosheets. The CoOOH nanosheet-loaded Au nanocluster material has both the fluorescence properties of AuNCs and the enzyme-mimicking catalytic properties of CoOOH nanosheets, and can be used for the ratio fluorescence detection of azodicarbonamide in flour.

[0035] Example 1: Preparation of AuNCs@CoOOH

[0036] Preparation method of CoOOH nanosheets: 3.0mL of NaOH (1.0M) and 10.0mL of CoCl2 (10mM) were mixed and then sonicated for 2min. Then 500μL of NaClO (0.9M) was injected and sonicated for 15min. Centrifuged at 6,000rpm for 5min, the precipitate was washed with water several times and ions were removed. Finally, the CoOOH nanosheets were freeze-dried to obtain a brown-black powder (A Smartphone-Assisted Robust Sensing Platform for On-Site Quantitation of 2,4-Dichlorophenoxyacetic Acid Using Red Emissive Carbon Dots).

[0037] Preparation of AuNCs: An aqueous solution was prepared by mixing freshly prepared HAuCl4 (20 mM, 0.50 mL) and GSH (100 mM, 0.15 mL) at 25°C with 4.35 mL of ultrapure water. The reaction mixture was heated to 70°C and gently stirred (500 rpm) for 24 h to obtain (From Aggregation-Induced Emission of Au(I)-Thiolate Complexes to Ultrabright Au(0)@Au(I)-Thiolate Core-Shell Nanoclusters).

[0038] 1 mL of AuNCs (0.38 mg / mL) aqueous solution and 1 mL of CoOOH nanosheets (0.12 mg / mL) aqueous solution were stirred and reacted at 25°C for 30 min. After centrifugation at 1000 rpm for 20 min, the upper mother liquor was poured off, the solid product was washed three times with water, and then vacuum dried at 60°C to obtain AuNCs@CoOOH.

[0039] Example 2: Characterization of the morphology, fluorescence and enzyme-like catalytic performance of CoOOH nanosheet-loaded Au nanocluster materials

[0040] The AuNCs@CoOOH prepared in Example 1 was characterized by transmission electron microscopy. Figure 1 As shown in A, AuNCs@CoOOH presents regular hexagonal flakes with a particle size of about 50-80nm. Figure 1 As shown in B, the loaded AuNCs can be observed with a particle size of about 2-3 nm and uniform distribution. Figure 1 C scanning transmission electron microscopy image shows that AuNCs (white dots) are uniformly dispersed on the CoOOH nanosheet support. Figure 1The D-1I elemental mapping showed that Co, Au, C, N, O, and S elements were uniformly distributed on the support, confirming the successful preparation of AuNCs@CoOOH.

[0041] The AuNCs@CoOOH prepared in Example 1 was characterized by fluorescence and enzyme-like catalytic performance. Figure 2 As shown in A, AuNCs has a characteristic fluorescence emission peak at 605nm. After forming AuNCs@CoOOH, the intensity of the characteristic fluorescence peak at 605nm decreases, which confirms that CoOOH nanosheets quench the fluorescence of AuNCs and that AuNCs@CoOOH has fluorescence properties. Figure 2 As shown in Figure B, AuNCs@CoOOH can catalyze the colorless substrates 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) to generate products with characteristic colors and UV absorption. Figure 2 As shown in Figure C, AuNCs@CoOOH can catalyze the oxidation of non-fluorescent thiamine into fluorescent thiochrome, confirming the enzyme-like catalytic properties of AuNCs@CoOOH.

[0042] Example 3: Azodicarbonamide ratio fluorescence detection based on CoOOH nanosheets loaded with Au nanoclusters

[0043] The AuNCs@CoOOH (0.13 mg / mL) prepared in Example 1 was dispersed and added into a mixture containing different concentrations of azodicarbonamide aqueous solution (0 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM), glutathione aqueous solution (60 μM) and thiamine aqueous solution (500 μM). The mixture was reacted at 60°C for 40 min, with an excitation wavelength of 365 nm to obtain a 605 nm (F 605 )、438nm(F 438 ) at the fluorescence intensity. With ADC concentration as the horizontal axis and fluorescence intensity ratio (F 605 / F 438 ) as the vertical axis to draw the standard curve.

[0044] Test results such as Figure 3 As shown in A, as the ADC content increases from 0 to 100 μM, the fluorescence emission intensity of the detection system at 605 nm decreases, and the fluorescence emission intensity at 438 nm increases. Figure 3 B is the fluorescence intensity ratio (F 605 / F 438 ) and the fitting straight line F between the ADC concentration 605 / F 438 =2.9688+0.0501[ADC], indicating that the detection range of this method for ADC is 0.1-50 μM, and the detection limit is 0.034 μM, which has an excellent detection effect.

[0045] Example 4: Selectivity of azodicarbonamide ratiometric fluorescence detection based on CoOOH nanosheets loaded with Au nanoclusters

[0046] The AuNCs@CoOOH (0.13 mg / mL) prepared in Example 1 was dispersed and the mixture was added with different ions (Na + , K + Mg 2+ NH4 + , Cl - 、SO4 2- 、NO3 - ), amino acids (lysine, tyrosine, glycine, valine, alanine, serine, arginine, aspartic acid, proline), sugars (glucose, sucrose, galactose, cyclodextrin, starch), other molecules (amylase, zein, adenosine, urea, cholesterol) aqueous solution, where cyclodextrin, starch, amylase and zein concentrations are 50 μg / mL, other ions and molecules concentrations are 50 μM, then add glutathione aqueous solution (60 μM) and thiamine aqueous solution (500 μM) to the above mixture, react at 60 ° C for 40 minutes. With 365 nm as the excitation wavelength, record 605 nm (F 605 )、438nm(F 438 ) is the fluorescence intensity at .

[0047] Test results such as Figure 4 As shown, the fluorescence intensity ratio is significantly reduced only in the presence of ADC, and there is no obvious change in the presence of other types of substances, which indicates that the sensing system has a high selectivity for ADC.

[0048] Example 5: Detection of azodicarbonamide in actual samples

[0049] The AuNCs@CoOOH (0.13 mg / mL) prepared in Example 1 was dispersed and added to commercial flour aqueous solutions containing different spiked concentrations. Then, glutathione aqueous solution (60 μM) and thiamine aqueous solution (500 μM) were added to the above mixtures and reacted at 60°C for 40 min. The excitation wavelength was 365 nm and the fluorescence intensity at 605 nm (F 605 )、438nm(F 438 ) and calculate the fluorescence intensity ratio, and substitute the ratio into the standard curve to calculate the content of ADC in the actual sample to be tested. The detected data results are shown in Table 1.

[0050] The recovery rate of ADC was 98.95%-100.91%, and the relative standard deviation was less than 4.17%. The results showed that the ratiometric fluorescence sensing method has high accuracy and reliability and can be used for the analysis and detection of ADC in actual samples.

[0051] Table 1 Detection results of ADC in actual samples

[0052]

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the ratio fluorescence of azodicarbonamide based on CoOOH nanosheets loaded with Au nanocluster materials, characterized in that: The CoOOH nanosheet-loaded Au nanocluster material comprises a CoOOH nanosheet and an Au nanocluster loaded on the CoOOH nanosheet; the detection method comprises the following steps: Step 1, preparing aqueous solutions containing different amounts of azodicarbonamide as standard solutions; Step 2, adding the CoOOH nanosheet loaded with Au nanocluster material into a mixed solution containing the azodicarbonamide standard solution prepared in step 1, and a glutathione aqueous solution and a thiamine aqueous solution, and performing fluorescence detection after the reaction.

2. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials according to claim 1, characterized in that: With 365±5 nm as the excitation wavelength, the fluorescence emission intensities at 605±5 nm and 438±5 nm were collected, and the relationship between the fluorescence intensity ratio at 605±5 nm and 438±5 nm and the concentration of azodicarbonamide was established to detect the concentration of azodicarbonamide.

3. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials according to claim 2, characterized in that: The particle size of the CoOOH nanosheet-loaded Au nanocluster material is 50-80 nm.

4. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials according to claim 3, characterized in that: The preparation method of CoOOH nanosheet-loaded Au nanocluster material is as follows: the AuNCs aqueous solution and the CoOOH nanosheet aqueous solution are mixed and stirred to obtain the material by a one-step electrostatic assembly method.

5. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials according to claim 4, characterized in that: In the preparation method, the mass concentration of the AuNCs aqueous solution is 0.3-0.5 mg / mL, and the mass concentration of the CoOOH nanosheet aqueous solution is 0.1-0.3 mg / mL.

6. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials according to claim 5, characterized in that: In the preparation method, the stirring reaction temperature is 20-35° C. and the reaction time is 25-40 min.

7. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials according to claim 6, characterized in that: In step 2, the mass concentration of the CoOOH nanosheet-loaded Au nanocluster material is 0.1-0.2 mg / mL, the concentration of the glutathione aqueous solution is 50-75 μM, and the concentration of the thiamine aqueous solution is 400-600 μM.

8. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials according to claim 7, characterized in that: In step 2, the reaction temperature is 45-65° C. and the reaction time is 25-45 min.

9. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials according to claim 8, characterized in that: The concentration range of azodicarbonamide detectable by the method is 0.1 to 50 μM.

10. Application of the azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanocluster materials according to any one of claims 1 to 9 in flour detection, characterized in that: In step 2, the concentration of azodicarbonamide is used as the horizontal axis and the fluorescence intensity ratio F 605 / F 438 A standard curve is drawn as the ordinate; CoOOH nanosheets loaded with Au nanocluster materials are prepared, and added to flour aqueous solutions containing different spiked concentrations, and then glutathione aqueous solution and thiamine aqueous solution are added to the above mixed solution, and the reaction is carried out at 60°C for 40 minutes; 365nm is used as the excitation wavelength, the fluorescence intensity at 605nm and 438nm is recorded, and the fluorescence intensity ratio is calculated, and the ratio is substituted into the standard curve to calculate the content of azodicarbonamide in the actual sample to be tested.

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