A method for detecting TBHQ based on fluorescence and colorimetric dual-mode signals of silver nanocluster nanozymes

Abstract

The present invention discloses a method for detecting TBHQ based on the fluorescence and colorimetric dual-mode signals of silver nanocluster nanozymes. The method comprises mixing a prepared silver nanocluster nanozyme Ag NCs solution with TBHQ in a buffer solution. The quinone-form product of TBHQ catalytically oxidized by the nanozyme reacts with the amino group modified by the nanozyme to form a red complex. The resulting characteristic absorption peak intensity and the corresponding fluorescence intensity change of the nanozyme are used for detection. A TBHQ working curve is prepared, and the TBHQ content in the sample is then determined based on the working curve. The method of the present invention uses polyethyleneimine as a stabilizer and formaldehyde as a reducing agent. Silver nanoclusters with excellent catalytic and fluorescent properties are prepared by adjusting the silver ion concentration. Based on the fluorescence / colorimetric dual-mode signal response formed by the oxidation product of TBHQ and the polyethyleneimine-stabilized silver nanoclusters, a new method for accurate and sensitive dual-mode detection of TBHQ 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 field of chemical analysis technology. Background Art

[0002] During food processing, storage, and cooking, oxidative deterioration and spoilage of edible oils can pose dangerous health risks to consumers. Compared to other synthetic antioxidants, tert-butylhydroquinone (TBHQ) has the advantages of strong antioxidant capacity, low cost, and easy availability. It is widely used as a food additive to maintain the nutritional and sensory qualities of edible oils and extend their shelf life. However, long-term exposure to high doses of TBHQ poses significant risks to public health and a range of side effects, including 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 addition amount of TBHQ should not exceed 200 mg / kg ("National Food Safety Standard: Standard for the Use of Food Additives (GB2760-2014), 2014). TBHQ has strong antioxidant properties 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 somewhat toxic to humans, the TBBQ produced by its decomposition during long-term storage in food becomes more biotoxic. Therefore, accurate and rapid detection of TBHQ is of paramount importance.

[0003] Numerous analytical methods have been developed for TBHQ detection, including high-performance liquid chromatography (HPLC), gas chromatography (GC), and electrochemical detection. While these methods are generally capable of accurately determining the TBHQ content in foods, they also have limitations. In particular, TBHQ may oxidize during food storage, leading to inaccurate content determination.

[0004] Nanozymes, alternatives to natural enzymes, are emerging as important tools in biosensing and analytical chemistry due to their unique catalytic activity and stability. They are typically made from metal nanoparticles (e.g., iron, copper, silver) or metal oxide nanoparticles (e.g., iron oxide, copper oxide). Nanozymes with oxidase-like activity have attracted considerable attention in the detection of antioxidants, effectively catalyzing their oxidation in the presence of oxygen. Therefore, we proposed a novel colorimetric / fluorimetric strategy for the oxidase-catalyzed oxidation of TBHQ (TBHQ) using a polyethylenimine-protected silver nanocluster nanozyme. This nanozyme catalyzes the oxidation of TBHQ in the presence of O₂ to produce the oxidation product, 2-tert-butyl-1,4-benzoquinone (TBBQ). Subsequently, a colorimetric reaction between TBBQ and imino groups modified on the surface of silver nanoclusters was exploited to produce a stable, colored product. Its effect on the fluorescence intensity of the silver nanoclusters can serve 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 colorimetric substrates or colorants are required during the detection process. At the same time, it utilizes a colorimetric / fluorescence dual-mode response, which 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] 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:

[0007] Step S1: Synthesis of Polyethyleneimine-Stabilized Silver Nanocluster Nanozymes

[0008] 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 sequence and mixed thoroughly to control the total volume to 10 mL; then stirred for 2 minutes to mix evenly, 96 μL of 35% formaldehyde solution was added under stirring conditions, and then stirred for 15 minutes. The resulting solution was centrifuged at 12,000 rpm for 10 minutes to remove large particles of silver nanoparticles, thereby obtaining a stable silver nanocluster nanozyme solution;

[0009] Step S2: Plotting the TBHQ concentration-fluorescence change standard curve

[0010] 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.

[0011] Step S3: Plotting the TBHQ concentration-absorbance standard curve

[0012] 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, and the absorbance at a wavelength of 504 nm was recorded. A standard working curve of TBHQ concentration-absorbance was drawn.

[0013] Step S4: Determination of TBHQ oxidation product spectrum measurement parameters

[0014] 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 minutes before performing UV-visible absorption and fluorescence spectroscopic measurements.

[0015] Step S5: Determination of TBHQ content in the sample to be tested

[0016] Replace the TBHQ solution in step S2 with the test solution and repeat step S2 until the change in fluorescence intensity at a wavelength of 450 nm is recorded. The change value is compared with the TBHQ concentration-fluorescence intensity change standard working curve obtained in step S2 to obtain the TBHQ concentration of the test solution;

[0017] 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.

[0018] Preferably, the concentration of the TBHQ standard solution in step S4 is 10-1000 μM.

[0019] Preferably, the buffer solution is a phosphate buffer solution with a pH value of 8.2.

[0020] 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, there is a good linear relationship between the concentration of TBHQ and the absorption peak intensity at 504 nm, and 504 nm is selected as the UV-visible absorption spectrum detection parameter of TBHQ.

[0021] Preferably, the fluorescence spectrum in step S4 is measured as follows: with an excitation wavelength of 380 nm, 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 fluorescence intensity change of the TBHQ solution in the concentration range of 5-400 μM and the Ag NCs nanozyme solution at a wavelength of 450 nm shows a good linear relationship, and 450 nm is selected as the TBHQ fluorescence spectrum detection parameter.

[0022] Preferably, in step S5, standard curves are drawn with TBHQ concentration as the horizontal axis and absorbance intensity and fluorescence intensity change values ​​as the vertical axis to obtain a linear regression equation.

[0023] 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. The measured values ​​are respectively substituted into the linear regression equation to calculate the TBHQ content in the sample.

[0024] Preferably, the sample extract is prepared by mixing 1-2 g of 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.

[0025] 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 combining the extract, which is filtered through a 0.45 μM membrane filter and then subjected to spectral analysis.

[0026] The present invention utilizes polyethyleneimine (PEI) as a stabilizer to produce a silver nanocluster nanozyme system (PEI-Ag NCs). Due to its superior catalytic properties, PEI selectively oxidizes TBHQ to form the quinone oxide TBBQ. The specific colorimetric reaction between TBBQ and the silver nanocluster-surface-modified PEI generates a red compound with a maximum absorption at 504 nm.

[0027] On the other hand, the connection between the amino groups of PEI and TBBQ produces a quenching effect on the fluorescence properties of the silver nanoclusters. Based on the above color development and fluorescence quenching reactions, a fluorescence / colorimetric dual-mode detection method for TBHQ in edible oils was established. Compared with the traditional direct detection of TBHQ, the present invention detects TBHQ after complete oxidation, which has higher accuracy. Furthermore, the present invention utilizes dual-mode signal detection, which has the advantages of a wide linear detection range and high sensitivity. Our research provides a new approach and strategy for the accurate and rapid detection of food antioxidants. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art.

[0029] Figure 1 Transmission electron microscopy image of the prepared PEI-Ag NCs and the size distribution of nanoclusters;

[0030] Figure 2 UV absorption and fluorescence spectra of the prepared PEI-Ag NCs;

[0031] Figure 3 The effect diagram of the prepared PEI-Ag NCs catalyzing the oxidation of TBHQ (the inset is a physical picture of the system, samples 1 to 5 are TBHQ, Ag+TBHQ, Ag, Ag+TBBQ, and TBBQ solutions);

[0032] Figure 4 UV-visible absorption spectra of TBHQ catalytic oxidation by PEI-Ag NCs under different pH conditions;

[0033] Figure 5 The UV-visible absorption spectra of the TBHQ oxidation system catalyzed by PEI-Ag NCs at different nanozyme dosages;

[0034] Figure 6 The maximum absorbance value change trend diagram of the TBHQ oxidation system catalyzed by PEI-Ag NCs under different time conditions;

[0035] 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 450 nm and the TBHQ concentration after adding different concentrations of TBHQ;

[0036] Figure 8 is the fluorescence spectrum of the PEI-Ag NCs system; Figure 8 A is the fluorescence spectra 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 fluorescence intensity change at a wavelength of 450 nm corresponding to the TBHQ concentration after adding different concentrations of TBHQ. DETAILED DESCRIPTION

[0037] The method for detecting TBHQ based on the fluorescence and colorimetric dual-mode signals of silver nanocluster nanozymes of the present invention comprises the following steps:

[0038] Step S1: Synthesis of Polyethyleneimine-Stabilized Silver Nanocluster Nanozymes

[0039] 1mL of 10mmol·L -1 AgNO3 solution and 1.67 mL of 0.05 g mL -1 Polyethyleneimine was added to ultrapure water in sequence and mixed thoroughly to control the total volume to 10 mL; then, after stirring for 2 minutes to mix evenly, 96 μL of 35% formaldehyde solution was added under stirring conditions, and stirring was continued for 15 minutes. The resulting solution was centrifuged at 12,000 rpm for 10 minutes to remove large particles of silver nanoparticles, thereby obtaining a stable silver nanocluster nanozyme solution;

[0040] Step S2: Plotting the TBHQ concentration-fluorescence change standard curve

[0041] 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.

[0042] Step S3: Plotting the TBHQ concentration-absorbance standard curve

[0043] 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, and the absorbance at a wavelength of 504 nm was recorded to draw a TBHQ concentration-absorbance standard working curve;

[0044] Step S4: Determination of TBHQ oxidation product spectrum measurement parameters

[0045] 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 minutes before performing UV-visible absorption and fluorescence spectroscopic measurements.

[0046] Step S5: Determination of TBHQ content in the sample to be tested

[0047] Replace the TBHQ solution in step S2 with the test solution and repeat step S2 until the change in fluorescence intensity at a wavelength of 450 nm is recorded. The change value is compared with the TBHQ concentration-fluorescence intensity change standard working curve obtained in step S2 to obtain the TBHQ concentration of the test solution;

[0048] 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.

[0049] This invention utilizes polyethyleneimine (PEI) as a stabilizer to produce a silver nanocluster nanozyme system (PEI-AgNCs). Due to its superior catalytic properties, PEI selectively oxidizes TBHQ to form the quinone oxide TBBQ. The specific color reaction between TBBQ and the silver nanocluster-surface-modified PEI produces a red compound with a maximum absorption at 504 nm.

[0050] On the other hand, the connection between the amino groups of PEI and TBBQ produces a quenching effect on the fluorescence properties of the silver nanoclusters. Based on the above color development and fluorescence quenching reactions, a colorimetric / fluorescence dual-mode detection method for TBHQ in edible oils was established. Compared with the traditional direct detection of TBHQ, the present invention detects TBHQ after complete oxidation, which has higher accuracy. Furthermore, the present invention utilizes dual-mode signal detection, which has the advantages of a wide linear detection range and high sensitivity. Our research provides a new approach and strategy for the accurate and rapid detection of food antioxidants.

[0051] The concentration of the TBHQ standard solution in step S4 is 10-1000 μM; the buffer solution is a phosphate buffer solution with a pH of 8.2. The UV-visible absorption spectrum in step S4 is determined by generating a new absorption peak at 504 nm in the mixed solution after adding TBHQ. Within the concentration range of 10-1000 μM, a good linear relationship is observed between the concentration of TBHQ and its absorption peak intensity at 504 nm. 504 nm is selected as the UV-visible absorption spectrum detection parameter for TBHQ. The fluorescence spectrum in step S4 is determined by using 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 TBHQ solution. The fluorescence intensity changes at a wavelength of 450 nm between the TBHQ solution and the AgNCs nanozyme solution within the concentration range of 5-400 μM show a good linear relationship. 450 nm is selected as the TBHQ fluorescence spectrum detection parameter.

[0052] In step S5 above, standard curves were plotted with TBHQ concentration as the abscissa and absorbance and fluorescence intensity changes as the ordinates, respectively, to obtain a linear regression equation. The sample extract was pipetted, 750 μL of the 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 minutes before UV-visible absorption and fluorescence spectra were measured. The measured values ​​were substituted into the linear regression equation to calculate the TBHQ content in the sample. The sample extract was prepared by mixing 1-2 g of the oil sample with 2-4 mL of methanol, sonicating for 5-30 minutes, and centrifuging at 4000-5000 rpm for 5 minutes to obtain a supernatant. The supernatant extraction process was repeated twice, and a total volume of 5-10 mL of supernatant extract was obtained. This extract was filtered through a 0.45 μM membrane filter and then subjected to spectral analysis.

[0053] The present invention will be further described below with reference to four embodiments. Example 1

[0054] The synthesis of polyethyleneimine-modified silver nanocluster nanozymes (PEI-Ag NCs) was carried out as follows:

[0055] 1 mL of 10 mmol·L -1 AgNO3 solution and 1.67 mL 0.05 g mL -1 Polyethyleneimine (PEI) was added sequentially to ultrapure water and thoroughly mixed, ultimately achieving a total volume of 10 mL. After stirring for 2 minutes to ensure uniform mixing, 96 μL of 35% formaldehyde solution was added while stirring. Stirring was continued for 15 minutes, and the resulting solution was centrifuged at 12,000 rpm for 10 minutes to remove larger silver nanoparticles, resulting in a stable, light yellow silver nanocluster nanozyme solution (PEI-Ag NCs).

[0056] Depend on Figure 1 It can be seen that the average size of PEI-Ag NCs nanozymes is 2~5nm; its UV absorption spectrum and fluorescence spectrum are as follows Figure 2 shown.

[0057] Example 2: Test of catalytic and color development ability of PEI-Ag NCs nanozymes

[0058] (1) Synthesis of PEI-Ag NCs nanozymes: The synthesis method is the same as that in Example 1.

[0059] (2) To five 5 mL centrifuge tubes, add 750 μL of the PEI-Ag NCs solution prepared in Example 1, 1200 μL of 1 mM TBHQ standard solution, a mixture of 750 μL of PEI-Ag NCs solution and 1200 μL of 1 mM TBHQ standard solution, 1200 μL of 1 mM TBBQ standard solution, and a mixture of 750 μL of PEI-Ag NCs solution and 1200 μL of 1 mM TBBQ standard solution. Then, add 20 mM phosphate buffer solution (pH 8.2) to the tubes to bring the total volume of the system to 3000 μL. After mixing at room temperature for 25 min, measure the UV-visible absorption spectra of the systems.

[0060] 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. However, the mixed solutions of PEI-Ag NCs and TBHQ and PEI-Ag NCs and TBBQ all produced 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.

[0061] The imino group has a certain degree of reactivity and can participate in the formation of Schiff base compounds, that is, the imino group reacts with aldehydes or ketones to form covalent couplings. 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 then reacts with the carbonyl group of TBBQ to form a colored covalent compound.

[0062] Example 3: A colorimetric and fluorescence dual-mode detection method for TBHQ based on PEI-Ag NCs nanozymes, the specific steps are as follows:

[0063] Synthesis of PEI-Ag NCs nanozymes: The synthesis method is the same as that in Example 1;

[0064] Drawing of TBHQ-absorbance standard working curve:

[0065] In a 5 mL centrifuge tube, add 750 μL of the synthesized PEI-Ag NCs nanozyme, then add TBHQ solution of varying concentrations (10-1000 μM), and finally add 20 mM phosphate buffer solution (pH 8.2) to control the total volume to 3000 μL. Mix well and react at room temperature for 30 min. Figure 4As shown, the reaction system selected a phosphate buffer solution with a pH of 8.2. Figure 5 As shown in Figure 2, the amount of nanozyme added to the reaction system is 750 μL. Figure 6 As shown, the reaction time was 30 min.

[0066] The UV absorption spectrum of the mixed solution after color change was measured using a UV-visible absorption spectrometer, and the absorbance at a wavelength of 504 nm was recorded. Figure 7 A TBHQ concentration-absorbance standard working curve was plotted as shown. The absorbance of the mixed solution at 504 nm exhibited a good linear relationship with TBHQ concentration within two concentration ranges (10-300 μM and 300-1000 μM), with the resulting linear regression equations being y = 0.02277 + 0.0023x (R = 0.9973) and y = 0.00289x - 0.00512 (R = 0.9902), respectively.

[0067] Plotting of the working curve of the fluorescence intensity change of PEI-Ag NCs nanozyme and TBHQ concentration standard

[0068] To a 5 mL centrifuge tube, add 750 μL of the synthesized PEI-Ag NCs nanozyme, followed by TBHQ solutions of varying concentrations (5-400 μM), and finally 20 mM phosphate buffer (pH 8.2) to a final volume of 3000 μL. Mix thoroughly and react at room temperature for 30 min.

[0069] The fluorescence emission spectrum of the mixed solution after color change was measured by fluorescence spectrophotometer, 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).

[0070] Example 4: Determination of TBHQ content in edible oil

[0071] 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.

[0072] The drawing of the standard curve is the same as steps 2 and 3 in Example 2;

[0073] Sample Preparation: 2 g of edible oil sample was mixed with 4 mL of pure methanol in a 10 mL centrifuge tube. After sonication for 30 minutes, the extract was centrifuged at 5000 rpm for 5 minutes. The extract was then transferred to a 25 mL volumetric flask. This extraction process was repeated twice to ensure complete extraction. The entire extract was then collected, transferred to a 25 mL volumetric flask, and diluted to the mark with methanol.

[0074] The TBHQ content in the sample was determined by measuring the absorbance value at 504 nm and the fluorescence intensity change at 450 nm. The results were substituted into the linear regression equation in (1) and the TBHQ content in the edible oil was calculated to be 127.3 μM.

[0075] Recovery and precision experiments: Two different concentrations of TBHQ standard solutions were spiked into edible oil samples. Three replicates were performed for each concentration, and the spiked recoveries and relative standard deviations (RSDs) were calculated. The results are shown in Table 1. Detection of TBHQ in edible oil using PEI-AgNCs nanozymes. The spiked recoveries of TBHQ were measured to be between 95.7% and 101.2%, with RSDs between 1.7% and 3.5%. This method demonstrates good accuracy and precision.

[0076] Table 1 Detection of TBHQ content in edible oil by PEI-Ag NCs nanozyme

[0077] 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%

[0078] 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 this application should not be determined with reference to the above description, but rather 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 from the foregoing claims is not a disclaimer of such subject matter, nor should it be assumed that the inventors did not consider such subject matter to be part of the disclosed inventive subject matter.

Claims

1. A method for detecting TBHQ based on the dual-mode fluorescence and colorimetric signals of silver nanocluster nanozymes, characterized by: 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 sequence and mixed thoroughly to control the total volume to 10 mL; then, after stirring for 2 minutes to mix evenly, 96 μL of 35% formaldehyde solution was added under stirring conditions, and then stirring was continued for 15 minutes. The resulting solution was centrifuged at 12,000 rpm for 10 minutes to remove large particles of silver nanoparticles, thereby obtaining 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, and the absorbance at a wavelength of 504 nm was recorded. A standard working curve of TBHQ concentration-absorbance was drawn. Step S4: Determination of TBHQ oxidation product spectrum measurement parameters 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 before performing UV-visible absorption and fluorescence spectroscopic measurements, respectively. Step S5: Determination of TBHQ content in the sample to be tested Replace the TBHQ solution in step S2 with the test solution and repeat step S2 until the change in fluorescence intensity at a wavelength of 450 nm is recorded. The change value is compared with the TBHQ concentration-fluorescence intensity change standard working curve obtained in step S2 to obtain the TBHQ concentration of the test solution; Replace the TBHQ solution in step S3 with the test solution and repeat step S3 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 according to 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 measurement in step S4 is 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 the absorption peak intensity at 504 nm. 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 according to claim 1, characterized in that: The fluorescence spectrum measurement in step S4 is as follows: with an excitation wavelength of 380 nm, 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 step S5, standard curves are drawn with TBHQ concentration as the horizontal axis and absorbance intensity and fluorescence intensity change values ​​as the vertical axis to obtain a 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 according to claim 7, characterized in that: The sample extract is prepared by mixing 1-2 g of 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 according to 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 combining the extracts. The extracts were filtered through a 0.45 μM membrane filter and then subjected to spectral analysis.

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