A method for detecting the ratiometric fluorescence of azodicarbonamide based on CoOOH nanosheets loaded with Au nanoclusters

Abstract

The present invention discloses a method for detecting azodicarbonamide ratio fluorescence based on CoOOH nanosheet-loaded Au nanocluster materials, belonging to the field of fluorescence sensing technology. The CoOOH nanosheet-loaded Au nanocluster material provided by the present invention is obtained by subjecting AuNCs and CoOOH nanosheets to a one-step electrostatic assembly method. The CoOOH nanosheet-loaded Au nanocluster material has both the fluorescence properties of AuNCs and the enzyme-mimicking catalytic properties of CoOOH nanosheets. The present invention also provides a method for detecting azodicarbonamide ratio fluorescence based on CoOOH nanosheet-loaded Au nanocluster materials. Compared with existing detection methods, the method has the following significant advantages: the method has high selectivity; the method utilizes the ratio fluorescence mechanism to effectively reduce the interference caused by environmental and instrument fluctuations, thereby achieving reliable quantitative analysis of azodicarbonamide, and has the advantages of high sensitivity and high credibility, and has broad prospects in the field of fluorescence sensing detection.

Description

Technical Field

[0001] The present 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 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 those skilled in the art.

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

[0004] Common methods for detecting azodicarbonamide (AZO4) in flour include high-performance liquid chromatography (HPLC), fluorescence colorimetry, spectrophotometry, infrared spectroscopy, hyperspectral imaging, terahertz time-domain spectroscopy, and Raman spectroscopy (Tian Linshuang et al., "Research Progress on Detection Methods of Azodicarbonamide in Wheat Flour and Its Products"). Traditional chemical detection methods offer high precision but low detection limits. However, some methods are time-consuming, cumbersome, and sample-damaging, limiting their use to random inspections by quality inspection departments and preventing the detection of all agricultural products. Fluorescence analysis has attracted attention due to its ease of use and cost-effectiveness. Currently proposed fluorescence methods rely on changes in a single fluorescence signal and can be susceptible to interference from uncontrollable factors such as probe concentration, photobleaching, and changes in environmental conditions. To overcome the challenges faced by single-emission fluorescence methods, ratiometric fluorescence is the most effective strategy. Ratiometric fluorescence offers self-calibration and high selectivity, resulting in excellent performance in sensing and detection.

[0005] CoOOH nanosheets, as two-dimensional hexagonal transition metal oxyhydroxide nanomaterials, have shown great potential in molecular detection. The main advantage of CoOOH nanosheets over other 2D nanomaterials is that they can be prepared under mild conditions in a short time without the need for expensive instruments. CoOOH nanosheets can act as effective quenchers to quench fluorescent molecules adsorbed on their surfaces. Chu et al. first developed a CoOOH-modified upconversion nanosystem, utilizing 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 (such as van der Waals forces and lipophilic interactions) and increased the restriction of intramolecular vibration and rotation of the complex, thereby enhancing the catalytic activity of AuNCs, thereby achieving the reduction of Pb 2+ Analytical testing.

[0007] The patent publication, "A Fluorescence Immunoassay Method for Imidacloprid Based on Gold Nanoclusters Anchored Cobalt Oxyhydroxide Nanosheets" (Publication No. CN109580939A), discloses the use of glutathione-functionalized gold nanoclusters (AuNCs) and cobalt oxyhydroxide nanosheets (CoOOH NPs) to prepare a CoOOH-AuNCs composite for the detection of imidacloprid. However, the feasibility of this technique for the detection of azodicarbonamide in flour remains uncertain. Summary of the Invention

[0008] In response to the shortcomings of existing technologies, this paper provides a ratiometric fluorescence detection method for azodicarbonamide based on CoOOH nanosheets loaded with Au nanoclusters. This method utilizes AuNCs@CoOOH, a material with both fluorescent and enzyme-mimicking catalytic properties. The resulting ratiometric fluorescence sensing method, developed based on AuNCs@CoOOH, is capable of detecting azodicarbonamide with high sensitivity, 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: Add the CoOOH nanosheet loaded with Au nanocluster material into a mixture of the azodicarbonamide standard solution prepared in step 1, a glutathione aqueous solution, and a thiamine aqueous solution, and perform fluorescence detection after the reaction.

[0013] Preferably, the excitation wavelength is 365±5 nm, 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 azodicarbonamide concentration is established to detect the azodicarbonamide concentration.

[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 then 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 ratiometric fluorescence detection method provided by the present invention has the advantages of a wide linear range (0.1 to 50 μM), a detection limit as low as 0.034 μM (3.95 ppb), and simple operation for the detection of azodicarbonamide.

[0022] In the 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 was drawn on the vertical axis; CoOOH nanosheets loaded with Au nanocluster materials were prepared and added to flour aqueous solutions containing different spiked concentrations, and then glutathione aqueous solution and thiamine aqueous solution were added to the above mixtures, and the reaction was carried out at 60°C for 40 minutes; with 365nm as the excitation wavelength, the fluorescence intensity at 605nm and 438nm was recorded, and the fluorescence intensity ratio was calculated. The ratio was 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; CoOOH nanosheets quench the fluorescence of AuNCs through fluorescence resonance energy transfer (FRET); in addition, CoOOH nanosheets have oxidase-like properties and can catalyze the oxidation of non-fluorescent thiamine into fluorescent thiochromes.

[0025] (2) The ratiometric fluorescence detection method for azodicarbonamide based on CoOOH nanosheets loaded with Au nanoclusters provided by the present invention has significant advantages over the existing detection methods for azodicarbonamide in flour: the method has high selectivity; the use of the ratiometric fluorescence mechanism can effectively reduce the interference caused by environmental and instrument fluctuations, and realize reliable quantitative analysis of azodicarbonamide. It has the advantages of high sensitivity and high reliability, and has broad prospects in the field of fluorescence sensing detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, 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), scanning transmission electron microscopy image (C), and Co, Au, C, N, O and S elemental mapping (DI).

[0028] Figure 2 Fluorescence emission spectra of AuNCs and AuNCs@CoOOH in Example 2 (A), UV absorption spectra and visible light photographs of AuNCs@CoOOH before and after catalysis of chromogenic substrates (B), and fluorescence emission images 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 solution in Example 3 are shown. 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 Schematic diagram of the mechanism of azodicarbonamide detection of CoOOH nanosheet-loaded Au nanocluster materials synthesized in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as 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 with reference to 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. Figure 5 As shown, the CoOOH nanosheet-loaded Au nanocluster material was prepared by a one-step electrostatic assembly of AuNCs and CoOOH nanosheets. This CoOOH nanosheet-loaded Au nanocluster material combines the fluorescent properties of AuNCs with the enzyme-mimicking catalytic properties of CoOOH nanosheets and can be used for the ratiometric fluorescence detection of azodicarbonamide in flour.

[0035] Example 1: Preparation of AuNCs@CoOOH

[0036] CoOOH nanosheets were prepared by mixing 3.0 mL of 1.0 M NaOH and 10.0 mL of 10 mM CoCl₂, followed by sonication for 2 minutes. 500 μL of 0.9 M NaClO was then added and sonicated for another 15 minutes. The mixture was centrifuged at 6,000 rpm for 5 minutes, and the precipitate was washed multiple times with water to remove ions. Finally, the CoOOH nanosheets were freeze-dried to yield 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] AuNCs were 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 hours 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, and the solid product was washed three times with water and then dried in vacuum at 60°C to obtain AuNCs@CoOOH.

[0039] Example 2: Characterization of the morphology, fluorescence and enzyme-like catalytic properties of CoOOH nanosheets loaded with Au nanoclusters

[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 As shown in the C scanning transmission electron microscopy image, AuNCs (white dots) are uniformly dispersed on the CoOOH nanosheet support. Figure 1The D-II elemental mapping shows that Co, Au, C, N, O, and S elements are 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 Figure A, AuNCs has a characteristic fluorescence emission peak at 605nm. After forming AuNCs@CoOOH, the intensity of the fluorescence characteristic 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'-azinobis(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 ratiometric 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 ) fluorescence spectrum. 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 increased from 0 to 100 μM, the fluorescence emission intensity of the detection system at 605 nm decreased, and the fluorescence emission intensity at 438 nm increased. 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 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 containing 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), and other molecules (amylase, zein, adenosine, urea, cholesterol) in aqueous solution, where the concentrations of cyclodextrin, starch, amylase, and zein were 50 μg / mL, and the concentrations of other ions and molecules were 50 μM. Glutathione aqueous solution (60 μM) and thiamine aqueous solution (500 μM) were then added to the mixture and reacted at 60°C for 40 minutes. The excitation wavelength was 365 nm, and the fluorescence was recorded at 605 nm (F 605 )、438nm(F 438 ) is the fluorescence intensity at .

[0047] Test results such as Figure 4 As shown in the figure, 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 shows that the sensing system has 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 mixture 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. Substituting the ratio into the standard curve can calculate the ADC content in the actual sample to be tested. The test data results are detailed in Table 1.

[0050] The ADC recovery rate ranged from 98.95% to 100.91%, with a relative standard deviation of less than 4.17%. The results showed that this 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 foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

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

2. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanoclusters according to claim 1, characterized in that: With an excitation wavelength of 365±5 nm, the fluorescence emission intensities at 605±5 nm and 438±5 nm were collected, and the relationship between the ratio of the fluorescence intensities at 605±5 nm to 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 nanoclusters 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 nanoclusters 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, and the mixture is obtained by a one-step electrostatic assembly method.

5. The azodicarbonamide ratio fluorescence detection method based on CoOOH nanosheets loaded with Au nanoclusters 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 nanoclusters 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 nanoclusters 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 nanoclusters 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 nanoclusters 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 nanoclusters 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 is used as the horizontal axis. 605 / F 438 A standard curve was drawn on the vertical axis; CoOOH nanosheets loaded with Au nanocluster materials were prepared and added to flour aqueous solutions containing different spiked concentrations, and then glutathione aqueous solution and thiamine aqueous solution were added to the above mixtures, and the reaction was carried out at 60°C for 40 minutes; with 365nm as the excitation wavelength, the fluorescence intensity at 605nm and 438nm was recorded, and the fluorescence intensity ratio was calculated. The ratio was substituted into the standard curve to calculate the content of azodicarbonamide in the actual sample to be tested.

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