Method for detecting TBHQ based on silver nano-cluster nano-enzyme fluorescence and colorimetric dual-mode signals

Abstract

The invention discloses a method for detecting TBHQ (tert-butylhydroquinone) based on silver nano-cluster nano-enzyme fluorescence and colorimetric dual-mode signals, which comprises the following steps: mixing a prepared silver nano-cluster nano-enzyme Ag NCs solution and TBHQ in a buffer solution, generating a red complex from a quinonoid product of the TBHQ after catalytic oxidation of the nano-enzyme and amino modified by the nano-enzyme, and detecting the TBHQ through fluorescence and colorimetric dual-mode signals. Detecting by utilizing the intensity of the generated characteristic absorption peak and the change of the fluorescence intensity corresponding to the nano-enzyme, making a TBHQ working curve, and determining the TBHQ content in the sample according to the working curve. According to the method, polyethyleneimine is used as a stabilizer, formaldehyde is used as a reducing agent, silver nano-clusters with excellent catalytic characteristics and fluorescence characteristics are prepared through reduction by adjusting the concentration of silver ions, and fluorescence / colorimetric dual-mode signal response is formed based on an oxidation product of TBHQ and the polyethyleneimine-stabilized silver nano-clusters. And a novel accurate and sensitive dual-mode TBHQ detection method is established.

Description

Technical Field

[0001] The present invention relates to a method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals, and specifically to a method for detecting TBHQ based on silver nanocluster nanozyme fluorescence / colorimetric dual-mode signals with good specificity and high detection sensitivity, belonging to the technical field of chemical analysis. Background Art

[0002] During food processing, storage and cooking, oxidative deterioration and spoilage of edible oils may pose dangerous health problems to consumers. Compared with other synthetic antioxidants, tert-butylhydroquinone (TBHQ) has the advantages of strong antioxidant capacity, low cost and easy availability, and is widely used as a food additive to maintain the nutritional and sensory quality of edible oils and extend their shelf life. However, long-term exposure to high doses of TBHQ will pose significant risks to public health and a series of side effects, such as cytotoxicity, genotoxicity, carcinogenicity and mutagenic effects. Due to its potential toxicity, the European Union and Japan have banned its use in some foods. China's national food safety standards stipulate that the maximum amount of TBHQ added shall not exceed 200 mg / kg ("National Food Safety Standard: Food Additives Usage Standard (GB2760-2014), 2014). TBHQ has a strong antioxidant effect and is easily oxidized and transformed. A recent study showed that 2-tert-butyl-1,4-benzoquinone (TBBQ) is the main component of TBHQ oxidation products. Although TBHQ is toxic to humans, long-term storage of food makes the TBBQ produced by TBHQ decomposition more biologically toxic. Therefore, accurate and rapid detection of TBHQ is of great significance.

[0003] Many analytical methods have been developed for TBHQ detection, including high performance liquid chromatography (HPLC), gas chromatography (GC), electrochemical detection, etc. However, these methods can usually accurately determine the content of TBHQ in food, but they also have certain limitations, especially TBHQ may be oxidized during food storage, resulting in inaccurate content determination.

[0004] Nanozymes are nanoenzymes that are alternatives to natural enzymes and are becoming important tools in the fields of biosensing and analytical chemistry due to their unique catalytic activity and stability. They are usually made of metal nanoparticles (such as iron, copper, silver, etc.) or metal oxide nanoparticles (such as iron oxide, copper oxide, etc.). Among them, nanozymes with oxidase-like activity have attracted much attention in the detection of antioxidants and can effectively catalyze the oxidation of antioxidants in the presence of oxygen. Therefore, we proposed a new colorimetric / fluorescent strategy for the oxidase-catalyzed oxidation of TBHQ based on polyethyleneimine-protected silver nanocluster nanozymes. The nanozyme can catalyze the oxidation of TBHQ in the presence of O2 to generate the oxidation product 2-tert-butyl-1,4-benzoquinone (TBBQ). Subsequently, the colorimetric reaction between TBBQ and the imino groups modified on the surface of silver nanoclusters produced a stable, colored product, and its effect on the fluorescence intensity of silver nanoclusters can be used as a specific indicator for the rapid and accurate detection of TBHQ. Compared with the currently reported methods, the silver nanocluster nanozymes used in this technology are simple to synthesize, and no additional chromogenic substrates or chromogenic agents are required during the detection process. At the same time, it utilizes the colorimetric / fluorescence dual-mode response, and has the advantages of good specificity and high detection sensitivity. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting TBHQ based on silver nanocluster nanozyme fluorescence / colorimetric dual-mode signals, which has the characteristics of good specificity and high detection sensitivity.

[0006] In order to solve the above technical problems, the technical solution of the present invention is: a method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals, the innovation of which is that the method comprises the following steps: Step S1: Synthesis of Polyethyleneimine-Stabilized Silver Nanocluster Nanozymes 1 mL of 10 mmol·L -1 AgNO3 solution and 1.67 mL of 0.05 g mL -1 Polyethyleneimine was added to ultrapure water in turn and mixed thoroughly to control the total volume to 10 mL; then stirred for 2 min to make it evenly mixed, 96 μL of 35% formaldehyde solution was added under stirring, and then stirred for 15 min, and the resulting solution was centrifuged at 12,000 rpm for 10 min to remove large silver nanoparticles to obtain a stable silver nanocluster nanozyme solution; Step S2: Plotting the TBHQ concentration-fluorescence change standard curve Silver nanocluster nanozyme solution, TBHQ solution of different concentrations, and buffer solution were added to a centrifuge tube in sequence and mixed evenly at room temperature. The fluorescence spectrum of the mixed solution was measured, and the change in fluorescence intensity at 450 nm and the standard working curve of TBHQ concentration were recorded. Step S3: Plotting the TBHQ concentration-absorbance standard curve Silver nanocluster nanozyme solution, TBHQ solution of different concentrations, and buffer solution were added to a centrifuge tube in sequence and mixed evenly at room temperature. The UV-visible absorption spectrum of the mixed solution was measured, the absorbance at a wavelength of 504 nm was recorded, and a TBHQ concentration-absorbance standard working curve was drawn; Step S4: Determination of Spectral Parameters of TBHQ Oxidation Products Add 0.5-1 mL of silver nanocluster nanozyme solution and 10 mM TBHQ standard solution to a stoppered colorimetric tube, dilute to 3 mL with pH 8.2 buffer solution, shake well and let stand for 20 min, then perform UV-visible absorption and fluorescence spectroscopic measurements respectively; Step S5: Determination of TBHQ content in the sample to be tested Substituting the TBHQ solution in step S2 with the solution to be tested and repeating step S2 until recording the change in fluorescence intensity at a wavelength of 450 nm, comparing the change value with the TBHQ concentration-fluorescence intensity change standard working curve obtained in step S2, to obtain the TBHQ concentration of the solution to be tested; The TBHQ solution in step S3 is replaced by the solution to be tested and step S3 is repeated until the absorbance value at a wavelength of 504 nm is recorded. The absorbance value is compared with the TBHQ concentration-absorbance standard working curve obtained in step S3 to obtain the TBHQ concentration of the solution to be tested.

[0007] Preferably, the concentration of the TBHQ standard solution in step S4 is 10-1000 μM.

[0008] Preferably, the buffer solution is a phosphate buffer solution with a pH value of 8.2.

[0009] Preferably, the UV-visible absorption spectrum in step S4 is measured as follows: a new absorption peak is generated at 504 nm in the mixed solution to which TBHQ is added, and within the concentration range of 10-1000 μM, a good linear relationship is shown between the concentration of TBHQ and its absorption peak intensity at 504 nm, and 504 nm is selected as the UV-visible absorption spectrum detection parameter of TBHQ.

[0010] Preferably, the fluorescence spectrum in step S4 is measured with 380 nm as the excitation wavelength, and the emission peak intensity at 450 nm in the fluorescence emission spectrum of the silver nanocluster nanozyme solution shows a quenching trend with the addition of TBHQ solution; the TBHQ solution in the concentration range of 5-400 μM shows a good linear relationship with the fluorescence intensity change of the Ag NCs nanozyme solution at a wavelength of 450 nm, and 450 nm is selected as the TBHQ fluorescence spectrum detection parameter.

[0011] Preferably, in step S5, standard curves are drawn with TBHQ concentration as the horizontal coordinate and absorbance intensity and fluorescence intensity change values ​​as the vertical coordinates to obtain a linear regression equation.

[0012] Preferably, the sample extract is pipetted, 750 μL of silver nanocluster solution is added, diluted to 3 mL with buffer solution, shaken, and allowed to stand for 25 min before UV-visible absorption and fluorescence spectra are measured, and the measured values ​​are respectively substituted into the linear regression equation to calculate the TBHQ content in the sample.

[0013] Preferably, the sample extract is prepared by mixing 1-2 g of the oil sample with 2-4 mL of methanol, ultrasonicating for 5-30 min, and centrifuging at 4000-5000 rpm for 5 min to obtain a supernatant.

[0014] Preferably, in the preparation of the sample extract, the supernatant extraction process is repeated twice, and the supernatant extract with a total volume of 5-10 mL is obtained by merging, and the extract is filtered through a 0.45 μM membrane filter and then subjected to spectral analysis.

[0015] Beneficial effects of the present invention: The present invention uses polyethyleneimine (PEI) as a stabilizer to prepare a silver nanocluster nanozyme system (PEI-Ag NCs). Due to its superior catalytic properties, TBHQ is selectively oxidized to quinone oxide TBBQ. Due to the specific color reaction between TBBQ and PEI modified on the surface of silver nanoclusters, a red compound with maximum absorption at 504 nm is generated.

[0016] On the other hand, due to the connection between the amino group of PEI and TBBQ, a quenching effect is produced on the fluorescence characteristics of the silver nanoclusters. Based on the above color development and fluorescence quenching reactions, a fluorescence / colorimetric dual-mode response detection method for detecting TBHQ in edible oils was established. Compared with the traditional idea of ​​directly detecting TBHQ, the present invention detects TBHQ after completely oxidizing it, which has higher accuracy. At the same time, the present invention adopts dual-mode signal detection, which has the advantages of wide linear detection range and high sensitivity. Our research provides a new idea and strategy for the accurate and rapid detection of food antioxidants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the implementation modes of the present invention or the technical solutions in the prior art, the drawings required for use in the description of the implementation modes or the prior art are briefly introduced below.

[0018] Figure 1 Transmission electron microscope image of the prepared PEI-Ag NCs and the size distribution of nanoclusters; Figure 2 UV absorption and fluorescence spectra of the prepared PEI-Ag NCs; Figure 3 The effect diagram of the prepared PEI-Ag NCs catalytic oxidation of TBHQ (the embedded picture is the actual picture of the system, samples 1 to 5 are TBHQ, Ag+TBHQ, Ag, Ag+TBBQ, TBBQ solution); Figure 4 The UV-visible absorption spectra of PEI-Ag NCs catalytic oxidation of TBHQ system under different pH conditions; Figure 5 The UV-visible absorption spectra of the PEI-Ag NCs catalytic oxidation of TBHQ system at different nanozyme dosages; Figure 6 The maximum absorbance value variation trend diagram of PEI-Ag NCs catalytic oxidation of TBHQ system under different time conditions; Figure 7 is the UV-visible absorption spectrum of the PEI-Ag NCs system; Figure 7 A is the UV-visible absorption spectrum of the PEI-Ag NCs system after adding different concentrations of TBHQ under optimized conditions; Figure 7 B is the linear relationship diagram of the absorbance at a wavelength of 450nm and the TBHQ concentration after adding different concentrations of TBHQ; Figure 8 is the fluorescence spectrum of the PEI-Ag NCs system; Figure 8 A is the fluorescence spectrum of the PEI-Ag NCs system after adding different concentrations of TBHQ under optimized conditions; Figure 8 B is a linear relationship diagram of the change in fluorescence intensity at a wavelength of 450 nm corresponding to the TBHQ concentration after adding different concentrations of TBHQ. DETAILED DESCRIPTION

[0019] The method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals of the present invention comprises the following steps: Step S1: Synthesis of Polyethyleneimine-Stabilized Silver Nanocluster Nanozymes 1mL of 10mmol·L -1 AgNO3 solution and 1.67 mL of 0.05 g mL -1 Polyethyleneimine was added into ultrapure water in turn and mixed thoroughly to control the total volume to 10 mL; then stirred for 2 min to make it evenly mixed, 96 μL of 35% formaldehyde solution was added under stirring, and then stirred for 15 min, and the resulting solution was centrifuged at 12,000 rpm for 10 min to remove large silver nanoparticles and obtain a stable silver nanocluster nanozyme solution; Step S2: Plotting the TBHQ concentration-fluorescence change standard curve Silver nanocluster nanozyme solution, TBHQ solution of different concentrations, and buffer solution were added to a centrifuge tube in sequence and mixed evenly at room temperature. The fluorescence spectrum of the mixed solution was measured, and the change in fluorescence intensity at 450 nm and the standard working curve of TBHQ concentration were recorded. Step S3: Plotting the TBHQ concentration-absorbance standard curve Silver nanocluster nanozyme solution, TBHQ solution of different concentrations, and buffer solution were sequentially added into a centrifuge tube and mixed evenly at room temperature. The UV-visible absorption spectrum of the mixed solution was measured, the absorbance at a wavelength of 504 nm was recorded, and a TBHQ concentration-absorbance standard working curve was drawn; Step S4: Determination of Spectral Parameters of TBHQ Oxidation Products Add 0.5-1 mL of silver nanocluster nanozyme solution and 10 mM TBHQ standard solution to a stoppered colorimetric tube, dilute to 3 mL with pH 8.2 buffer solution, shake well and let stand for 20 min, then perform UV-visible absorption and fluorescence spectrometry measurements respectively; Step S5: Determination of TBHQ content in the sample to be tested Substituting the TBHQ solution in step S2 with the solution to be tested and repeating step S2 until recording the change in fluorescence intensity at a wavelength of 450 nm, comparing the change value with the TBHQ concentration-fluorescence intensity change standard working curve obtained in step S2, to obtain the TBHQ concentration of the solution to be tested; The TBHQ solution in step S3 is replaced by the solution to be tested and step S3 is repeated until the absorbance value at a wavelength of 504 nm is recorded. The absorbance value is compared with the TBHQ concentration-absorbance standard working curve obtained in step S3 to obtain the TBHQ concentration of the solution to be tested.

[0020] The present invention uses polyethyleneimine (PEI) as a stabilizer to prepare a silver nanocluster nanozyme system (PEI-AgNCs). Due to its superior catalytic properties, TBHQ is selectively oxidized to quinone oxide TBBQ. Due to the specific color reaction between TBBQ and PEI modified on the surface of silver nanoclusters, a red compound with maximum absorption at 504nm is generated.

[0021] On the other hand, due to the connection between the amino group of PEI and TBBQ, a quenching effect is produced on the fluorescence characteristics of the silver nanoclusters. Based on the above color development and fluorescence quenching reactions, a colorimetric / fluorescence dual-mode response detection method for detecting TBHQ in edible oils was established. Compared with the traditional idea of ​​directly detecting TBHQ, the present invention detects TBHQ after completely oxidizing it, which has higher accuracy. At the same time, the present invention adopts dual-mode signal detection, which has the advantages of wide linear detection range and high sensitivity. Our research provides a new idea and strategy for the accurate and rapid detection of food antioxidants.

[0022] The concentration of the TBHQ standard solution in the above step S4 is 10-1000 μM; the buffer solution is a phosphate buffer solution with a pH value of 8.2. The ultraviolet-visible absorption spectrum in step S4 is measured as follows: a new absorption peak is generated at 504 nm in the mixed solution to which TBHQ is added, and within the concentration range of 10-1000 μM, the concentration of TBHQ and its absorption peak intensity at 504 nm show a good linear relationship, and 504 nm is selected as the ultraviolet-visible absorption spectrum detection parameter of TBHQ. The fluorescence spectrum in step S4 is measured as follows: with 380 nm as the excitation wavelength, the emission peak intensity at 450 nm in the fluorescence emission spectrum of the silver nanocluster nanozyme solution shows a quenching trend with the addition of the TBHQ solution; the TBHQ solution shows a good linear relationship with the fluorescence intensity change of the AgNCs nanozyme solution at a wavelength of 450 nm within the concentration range of 5-400 μM, and 450 nm is selected as the TBHQ fluorescence spectrum detection parameter.

[0023] In the above step S5, the standard curves are drawn with the TBHQ concentration as the horizontal axis and the absorbance intensity and fluorescence intensity change values ​​as the vertical axis to obtain the linear regression equation. The sample extract is pipetted, 750 μL of silver nanocluster solution is added, diluted to 3 mL with buffer solution, shaken, and UV-visible absorption spectrum and fluorescence spectrum are measured after standing for 25 min. The measured values ​​are respectively substituted into the linear regression equation to calculate the TBHQ content in the sample. The sample extract is prepared by mixing 1-2 g of oil sample with 2-4 mL of methanol, ultrasonically treating for 5-30 min, and centrifuging at 4000-5000 rpm for 5 min to obtain a supernatant. In the preparation of the sample extract, the supernatant extraction process is repeated twice, and the total volume of the supernatant extract of 5-10 mL is obtained by merging, and the extract is filtered through a 0.45 μM membrane filter, and then spectrally analyzed.

[0024] The present invention is further described below by means of four embodiments. Example 1

[0025] The synthesis of polyethyleneimine-modified silver nanocluster nanozymes (PEI-Ag NCs) was carried out as follows: 1 mL of 10 mmol·L -1 AgNO3 solution and 1.67 mL 0.05 g mL -1 Polyethyleneimine (PEI) was added to ultrapure water in sequence and mixed thoroughly, and the total volume was finally controlled to be 10 mL. After stirring for 2 min to make it evenly mixed, 96 μL of 35% formaldehyde solution was added under stirring conditions, and then continued to stir for 15 min. The resulting solution was centrifuged at 12,000 rpm for 10 min to remove larger silver nanoparticles to obtain a stable light yellow silver nanocluster nanozyme solution (PEI-Ag NCs).

[0026] Depend on Figure 1 It can be seen that the average size of PEI-Ag NCs nanozymes is 2-5 nm; its UV absorption spectrum and fluorescence spectrum are as follows Figure 2 shown.

[0027] Example 2: Test of catalytic and color development ability of PEI-Ag NCs nanozymes (1) Synthesis of PEI-Ag NCs nanozymes: The synthesis method is the same as that in Example 1. (2) Add 750 μL of the PEI-Ag NCs solution prepared in Example 1, 1200 μL of 1 mM TBHQ standard solution, 750 μL of PEI-Ag NCs solution + 1200 μL of 1 mM TBHQ standard solution mixture, 1200 μL of 1 mM TBBQ standard solution, and 750 μL of PEI-Ag NCs solution + 1200 μL of 1 mM TBBQ standard solution mixture to five 5 mL centrifuge tubes, and then add 20 mM phosphate buffer solution with a pH of 8.2 to the tubes to determine that the total volume of the system is 3000 μL. After mixing evenly at room temperature for 25 min, measure the UV-visible absorption spectra of the system in turn.

[0028] The results are as follows Figure 3 As shown in the figure, the individual PEI-Ag NCs, TBHQ and TBBQ solutions have no obvious absorption peaks in the 400-600 nm region, while the mixed solutions of PEI-Ag NCs and TBHQ and PEI-Ag NCs and TBBQ all have maximum absorption peaks at 504 nm. Figure 3 In the embedded figure, the colors of the products after the PEI-Ag NCs solution was mixed with TBHQ and TBBQ were wine red, which was consistent with the corresponding UV absorption spectrum characterization results.

[0029] The imino group has a certain reactivity and can participate in the formation of Schiff base compounds, that is, the imino group reacts with aldehydes or ketones to form covalent coupling. Silver nanoclusters have oxidative activity as nanozymes. Figure 3 The results show that PEI-Ag NC nanozyme has good catalytic oxidation ability, which can oxidize TBHQ into TBBQ, and the imino group modified on the surface of silver nanozyme can generate colored covalent compounds with the carbonyl group of TBBQ.

[0030] Example 3: A colorimetric and fluorescence dual-mode detection method of TBHQ based on PEI-Ag NCs nanozymes, the specific steps are as follows: Synthesis of PEI-Ag NCs nanozyme, the synthesis method is the same as Example 1; Drawing of TBHQ-absorbance standard working curve: Add 750 μL of synthesized PEI-Ag NCs nanozyme to a 5 mL centrifuge tube, then add TBHQ solution of different concentrations (10-1000 μM), and finally add 20 mM phosphate buffer solution (pH 8.2) to control the final total volume to 3000 μL. Mix well and react for 30 min at room temperature. Figure 4 As shown, the reaction system selected a phosphate buffer solution with a pH of 8.2. Figure 5 As shown, the amount of nanozyme added to the reaction system is 750 μL. Figure 6 As shown, the reaction time was 30 min.

[0031] The UV absorption spectrum of the mixed solution after color change was measured by UV-visible absorption spectrometer, and the absorbance at a wavelength of 504 nm was recorded as follows: Figure 7 As shown, the TBHQ concentration-absorbance standard working curve was drawn. The absorbance value of the mixed solution at 504 nm showed a good linear relationship with the TBHQ concentration in two concentration ranges (10-300 μM, 300-1000 μM), and the obtained linear regression equations were y=0.02277+0.0023x (R=0.9973) and y=0.00289x-0.00512 (R=0.9902), respectively.

[0032] Plotting of the standard working curve of the fluorescence intensity change of PEI-Ag NCs nanozyme and TBHQ concentration Add 750 μL of synthesized PEI-Ag NCs nanozyme to a 5 mL centrifuge tube, then add TBHQ solution of different concentrations (5-400 μM), and finally add 20 mM phosphate buffer solution (pH 8.2) to control the final total volume to 3000 μL. Mix well and react for 30 min at room temperature.

[0033] The fluorescence emission spectrum of the mixed solution after color change was measured by a fluorescence photometer, and the change of fluorescence intensity at 450 nm was recorded. Figure 8 As shown, a standard working curve of TBHQ concentration-fluorescence intensity change was drawn, y=4.887x+524.2 (R=0.9953).

[0034] Example 4: Determination of TBHQ content in edible oil In order to demonstrate the practicality of the invention, we selected edible oil as the test sample and used the prepared PEI-Ag NCs nanozyme to detect the TBHQ content in the edible oil.

[0035] The drawing of the standard curve is the same as steps 2 and 3 in Example 2; Sample preparation: 2 g of edible oil sample was mixed with 4 mL of pure methanol in a 10 mL centrifuge tube. After ultrasonic treatment for 30 min, centrifuge at 5000 rpm for 5 min. Then, the extract was transferred to a 25 mL volumetric flask. The extraction process was performed twice to ensure thorough extraction. Then all the extracts were collected, transferred to a 25 mL volumetric flask, and diluted to the scale mark with methanol.

[0036] The TBHQ content in the sample was determined by measuring the absorbance value at 504 nm and the fluorescence intensity change at 450 nm and substituting them into the linear regression equation in (1). The TBHQ content in the edible oil was calculated to be 127.3 μM.

[0037] Recovery and precision experiment: Two different concentrations of TBHQ standard solutions were added to the edible oil samples. Each concentration was measured three times in parallel, and the spike recovery was calculated, and the relative standard deviation RSD was calculated. The results are shown in Table 1. PEI-AgNCs nanozyme detection of TBHQ content in edible oil. The spike recovery of TBHQ was measured to be between 95.7% and 101.2%, and the RSD was between 1.7% and 3.5%. This method has good accuracy and precision.

[0038] Table 1 Detection of TBHQ content in edible oil by PEI-Ag NCs nanozyme sample Addition amount (μM) Measured amount (μM) Recovery rate% RSD% 1 0 127.3 —— —— 2 100 217.6 95.7 1.7% 3 200 331.2 101.2 3.5% It should be understood that the above description is for illustration and not for limitation. Many embodiments and many applications beyond the examples provided will be apparent to those skilled in the art upon reading the above description. Therefore, the scope of the present application should not be determined with reference to the above description, but should be determined with reference to the appended claims and the full scope of equivalents to which such claims are entitled. For the purpose of comprehensiveness, all articles and references, including disclosures of patent applications and publications, are incorporated herein by reference. The omission of any aspect of the subject matter disclosed herein in the preceding claims is not intended to be a waiver of the subject matter, nor should it be considered that the inventors did not consider the subject matter to be part of the disclosed inventive subject matter.

Claims

1. A method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals, characterized in that: The method comprises the following steps: Step S1: Synthesis of Polyethylenimine-stabilized Silver Nanocluster Nanozymes 1 mL of 10 mmol·L -1 AgNO3 solution and 1.67 mL of 0.05 g mL -1 Polyethyleneimine was added into ultrapure water in turn and mixed thoroughly to control the total volume to 10 mL; then stirred for 2 min to make it evenly mixed, 96 μL of 35% formaldehyde solution was added under stirring, and then stirred for 15 min, and the resulting solution was centrifuged at 12,000 rpm for 10 min to remove large silver nanoparticles and obtain a stable silver nanocluster nanozyme solution; Step S2: Plotting the TBHQ concentration-fluorescence change standard curve Silver nanocluster nanozyme solution, TBHQ solution of different concentrations, and buffer solution were added to a centrifuge tube in sequence and mixed evenly at room temperature. The fluorescence spectrum of the mixed solution was measured, and the change in fluorescence intensity at 450 nm and the standard working curve of TBHQ concentration were recorded. Step S3: Plotting the TBHQ concentration-absorbance standard curve Silver nanocluster nanozyme solution, TBHQ solution of different concentrations, and buffer solution were added to a centrifuge tube in sequence and mixed evenly at room temperature. The UV-visible absorption spectrum of the mixed solution was measured, the absorbance at a wavelength of 504 nm was recorded, and a TBHQ concentration-absorbance standard working curve was drawn; Step S4: Determination of Spectral Parameters of TBHQ Oxidation Products Add 0.5-1 mL of silver nanocluster nanozyme solution and 10 mM TBHQ standard solution to a stoppered colorimetric tube, dilute to 3 mL with pH 8.2 buffer solution, shake well and let stand for 20 min, then perform UV-visible absorption and fluorescence spectrometry measurements respectively; Step S5: Determination of TBHQ content in the sample to be tested Substituting the TBHQ solution in step S2 with the solution to be tested and repeating step S2 until recording the change in fluorescence intensity at a wavelength of 450 nm, comparing the change value with the TBHQ concentration-fluorescence intensity change standard working curve obtained in step S2, to obtain the TBHQ concentration of the solution to be tested; The TBHQ solution in step S3 is replaced by the test solution and step S3 is repeated until the absorbance value at a wavelength of 504 nm is recorded. The absorbance value is compared with the TBHQ concentration-absorbance standard working curve obtained in step S3 to obtain the TBHQ concentration of the test solution.

2. The method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals according to claim 1, characterized in that: The concentration of the TBHQ standard solution in step S4 is 10-1000 μM.

3. The method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals as claimed in claim 1, characterized in that: The buffer solution is a phosphate buffer solution with a pH value of 8.

2.

4. The method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals according to claim 1, characterized in that: The UV-visible absorption spectrum in step S4 is measured as follows: a new absorption peak is generated at 504 nm in the mixed solution to which TBHQ is added, and within the concentration range of 10-1000 μM, a good linear relationship is shown between the concentration of TBHQ and its absorption peak intensity at 504 nm, and 504 nm is selected as the UV-visible absorption spectrum detection parameter of TBHQ.

5. The method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals as claimed in claim 1, characterized in that: The fluorescence spectrum in step S4 is measured as follows: with 380 nm as the excitation wavelength, the emission peak intensity at 450 nm in the fluorescence emission spectrum of the silver nanocluster nanozyme solution shows a quenching trend as the TBHQ solution is added; The fluorescence intensity changes of TBHQ solution in the concentration range of 5-400 μM and Ag NCs nanozyme solution at a wavelength of 450 nm showed a good linear relationship, and 450 nm was selected as the TBHQ fluorescence spectrum detection parameter.

6. The method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals according to claim 1, characterized in that: In the step S5, the standard curves are drawn with the TBHQ concentration as the horizontal coordinate and the absorbance intensity and the fluorescence intensity change value as the vertical coordinate to obtain the linear regression equation.

7. The method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals according to claim 6, characterized in that: The sample extract was pipetted, 750 μL of silver nanocluster solution was added, and the solution was diluted to 3 mL with buffer solution. The solution was shaken and allowed to stand for 25 min before UV-visible absorption and fluorescence spectra were measured. The measured values ​​were respectively substituted into the linear regression equation to calculate the TBHQ content in the sample.

8. The method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals as claimed in claim 7, characterized in that: The sample extract is prepared by mixing 1-2 g of the oil sample with 2-4 mL of methanol, ultrasonicating for 5-30 min, and centrifuging at 4000-5000 rpm for 5 min to obtain a supernatant.

9. The method for detecting TBHQ based on silver nanocluster nanozyme fluorescence and colorimetric dual-mode signals as claimed in claim 8, characterized in that: In the preparation of the sample extract, the supernatant extraction process was repeated twice, and the supernatant extract with a total volume of 5-10 mL was obtained by merging, and the extract was filtered through a 0.45 μM membrane filter and then subjected to spectral analysis.

Images