A method for colorimetric / fluorescent dual-mode rapid detection of sodium dehydroacetate by peroxidase-like / laccase-like nanoscale enzyme

Abstract

The application discloses a kind of peroxidase / laccase-like nanoscale enzyme colorimetric / fluorescence dual-mode rapid detection sodium dehydroacetate method, the method with 1-hydroxybenzotriazole (HOBT) as organic ligand, select Cu and Zn two transition metals as central ion, Cu / Zn-MOF material is synthesized.The linear range of colorimetric mode is 0.067-12 μg / mL, and the detection limit is 0.020 μg / mL;The linear range of fluorescence mode is 0.01-22 μg / mL, and the detection limit is 0.041 μg / mL, the sensor is applied to the recovery experiment of DHA in food sample, the recovery rate is between 92.1%~106.4%, and RSD is not more than 5%, verify that the analysis method has good stability, reliability and reproducibility.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, specifically to a rapid method for the detection of sodium dehydroacetate using a dual-mode colorimetric / fluorescence method similar to a peroxidase / laccase nanoenzyme. Background Technology

[0002] Sodium dehydroacetate (DHA-S) is a food additive with bactericidal and bacteriostatic effects. It can be used as a preservative in food and food products to inhibit mold growth and extend shelf life. This substance is difficult for the human body to metabolize and excrete; excessive intake can accumulate in the body, causing adverse reactions such as vomiting, confusion, and liver and kidney damage. The National Food Safety Standard for the Use of Food Additives (GB 2760-2024), implemented on February 8, 2025, has banned the use of sodium dehydroacetate in starch products, bread, pastries, and baked fillings. Therefore, determining the sodium dehydroacetate content in food is of great significance to human health. Currently, reported methods for determining DHA-S include high-performance liquid chromatography (HPLC), gas chromatography (GC), and electrochemiluminescence immunoassay. Colorimetric and fluorescence methods are important in detection technology due to their simplicity, speed, and lack of the need for large instruments; however, colorimetric and fluorescence detection of DHA-S are rarely reported.

[0003] Metal-organic frameworks (MOFs) are supramolecular assemblies composed of metal ions and organic ligands. They are candidate materials that can selectively generate reactive oxygen species (ROS) and have high utilization of active free radicals. The doped metal-nitrogen-carbon (Me-NC) structure constructed from MOFs can promote the uniform distribution of heteroatoms within the material. This structure can enhance the synergistic effect between adjacent unbonded metal atoms. At the same time, the catalytic activity and the types of nanozymes can be regulated by selecting appropriate ligands and central ions. Summary of the Invention

[0004] This invention provides a rapid, dual-mode colorimetric / fluorescence detection method for sodium dehydroacetate using a peroxidase / laccase nanoenzyme. The method uses 1-hydroxybenzotriazole (HOBT) as the organic ligand and selects Cu and Zn as the central transition metals to synthesize Cu / Zn-MOF materials. Cu / Zn-MOF exhibits excellent laccase-like activity, oxidizing 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP) to form a red quinone imine, producing a characteristic absorption peak at 505 nm. Simultaneously, Cu / Zn-MOF possesses peroxidase-like activity, oxidizing o-phenylenediamine (OPD) to generate the fluorescent product DAP, producing a characteristic fluorescence emission peak at 564 nm. The introduction of sodium dehydroacetate (DHA-S) specifically inhibits the laccase-like and POD-like activities of Cu / Zn-MOF, resulting in a lighter red color and reduced fluorescence intensity. This constructs a rapid and highly sensitive dual-mode colorimetric-fluorescence DHA-S sensing detection method. The linear range of the colorimetric mode is 0.067-12 μg / mL, with a detection limit of 0.020 μg / mL; the linear range of the fluorescence mode is 0.01-22 μg / mL, with a detection limit of 0.041 μg / mL. When this sensor was applied to the spiked recovery experiment of DHA-S in food samples, the recovery rate was between 92.1% and 106.4%, with an RSD not exceeding 5%, verifying that the analytical method has good stability, reliability, and reproducibility. The method of this invention features high sensitivity, strong specificity, simple operation, and rapidity.

[0005] The present invention relates to a method for rapid detection of sodium dehydroacetate using a colorimetric / fluorescence dual-mode method based on a peroxidase / laccase nanoenzyme, as follows:

[0006] (1) Dissolve 0.080-0.100g CuCl2·2H2O in 10-15mL methanol, 1.00-1.20g 1-hydroxybenzotriazole in 10-15mL methanol, and 0.100-0.150g Zn(OAc)2·2H2O in 10-15mL methanol. Sonicate each solution for 5-10min to ensure complete dissolution. Slowly add the CuCl2·2H2O solution to the 1-hydroxybenzotriazole solution and stir for 5-10min. Then slowly add the Zn(OAc)2·2H2O solution and continue stirring at room temperature for 12-15h. Let stand for 5-10h, centrifuge, remove the solid, wash with ethanol 3-4 times, and vacuum dry to obtain Cu / Zn-MOF nanozyme.

[0007] (2) Add 2,4-dichlorophenol and 4-aminoantipyrine solution to pH 6.5 MES buffer solution, add Cu / Zn-MOF nanozyme and sodium dehydroacetate standard solution of different concentrations, mix well, incubate at room temperature for 20-30 min, measure absorbance at 505 nm wavelength, determine the linear relationship between sodium dehydroacetate concentration and absorbance value, and obtain regression equation;

[0008] (3) Add o-phenylenediamine and H2O2 solution to pH 6.5 MES buffer solution, add Cu / Zn-MOF nanozyme and sodium dehydroacetate standard solution of different concentrations, mix well, incubate at room temperature for 20-30 min, and determine the linear relationship between sodium dehydroacetate concentration and fluorescence intensity by measuring the change in fluorescence intensity at 564 nm emission wavelength when the excitation wavelength is 421 nm, and obtain the regression equation.

[0009] (4) The absorbance and fluorescence intensity of the sample solution to be tested are measured according to the methods in steps (2) and (3). The absorbance and fluorescence intensity are then substituted into the regression equation to obtain the concentration of sodium dehydroacetate in the sample solution to be tested.

[0010] The Cu / Zn-MOF nanozyme solution has a concentration of 1 mg / mL and an addition amount of 50-100 μL; the 2,4-dichlorophenol solution has a concentration of 20 mmol / L and an addition amount of 100-200 μL; the 4-aminoantipyrine solution has a concentration of 20 mmol / L and an addition amount of 100-200 μL; the o-phenylenediamine solution has a concentration of 20 mmol / L and an addition amount of 100-200 μL; and the H2O2 solution has a concentration of 50 mmol / L and an addition amount of 100-200 μL.

[0011] The centrifugation described in step (1) is performed at 5000-10000 r / min for 5-10 min.

[0012] Advantages and technical effects of the present invention:

[0013] 1. This invention is based on the strong coordination ability of nitrogen atoms in 1-hydroxybenzotriazole with Cu2+ and Zn2+, allowing Cu / Zn-MOF to be prepared at room temperature. Simultaneously, the copper active sites in the Cu / Zn-MOF structure, mimicking natural laccase, are highly dispersed via zinc ions and the MOF backbone, exhibiting higher catalytic activity and stability than natural laccase and peroxidase. Under neutral conditions, Cu / Zn-MOF displays excellent laccase-like and peroxidase-like activities, capable of oxidizing 2,4-dichlorophenol and 4-aminoantipyrine to generate a red quinone imine, and oxidizing o-phenylenediamine to generate the fluorescent product DAP. The introduction of sodium dehydroacetate specifically inhibits the laccase-like and peroxidase-like activities of Cu / Zn-MOF, resulting in a lighter red color and reduced fluorescence intensity. This constructs a rapid and highly sensitive colorimetric-fluorescence DHA-S dual-mode sensing detection method.

[0014] 2. The linear range of the sodium dehydroacetate colorimetric mode established in this invention is 0.067-12 μg / mL, with a detection limit of 0.020 μg / mL; the linear range of the fluorescence mode is 0.01-22 μg / mL, with a detection limit of 0.041 μg / mL. When this sensor was applied to the spiked recovery experiment of DHA-S in food samples, the recovery rate was between 92.1% and 106.4%, with an RSD of no more than 5%. This analytical method has good stability, reliability, and reproducibility, and also features high sensitivity, strong specificity, simple operation, and speed. Attached Figure Description

[0015] Figure 1 TEM image of the Cu / Zn-MOF nanozyme prepared in Example 1;

[0016] Figure 2 The image shows the XRD pattern of Cu / Zn-MOF in Example 1.

[0017] Figure 3 The image shows the FT-IR spectrum of Cu / Zn-MOF in Example 1.

[0018] Figure 4 The high-resolution XPS spectra of Cu2p(a) and Zn2p(b) of Cu / Zn-MOF in Example 1 are shown.

[0019] Figure 5 The UV-vis absorption spectra of Cu / Zn-MOF catalytic oxidation of TMB and H2O2 in Example 1 are shown.

[0020] Figure 6 The UV-vis absorption spectra of Cu / Zn-MOF catalytic oxidation of 2,4-DP and 4-AP in Example 1 are shown.

[0021] Figure 7This is a radical capture diagram of the Cu / Zn-MOF+2,4-DP+4-AP system in Example 1;

[0022] Figure 8 This is a free radical capture diagram of the Cu / Zn-MOF+OPD+H2O2 system in Example 1;

[0023] Figure 9 The effect of KSCN on the catalytic activity of Cu / Zn-MOF in Example 1;

[0024] Figure 10 The effect of EDTA on the catalytic activity of Cu / Zn-MOF in Example 1;

[0025] Figure 11 The linear range of DHA-S detection by colorimetric method in Example 1;

[0026] Figure 12 The linear range for the fluorescence method used to detect DHA-S in Example 1;

[0027] Figure 13 The results show the selective detection of DHA-S by the Cu / Zn-MOF+4-AP+2,4-DP system (a) and the Cu / Zn-MOF+H2O2+OPD system (b) in Example 1.

[0028] Figure 14 The anti-interference test results are for the Cu / Zn-MOF+4-AP+2,4-DP+DHA-SS system (a) and the Cu / Zn-MOF+H2O2+OPD+DHA-S system (b) in Example 1. Detailed Implementation

[0029] The technical solution of the present invention will be described in further detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0030] Example 1: Determination of sodium dehydroacetate in food

[0031] 1. Preparation of Cu / Zn-MOF nanozymes: Dissolve 0.080g CuCl2·2H2O in 10mL methanol, 1.00g 1-hydroxybenzotriazole in 10mL methanol, and 0.100g Zn(OAc)2·2H2O in 10mL methanol. Sonicate each solution for 10min to ensure complete dissolution. Slowly add the CuCl2·2H2O solution to the 1-hydroxybenzotriazole solution and stir for 10min. Then slowly add the Zn(OAc)2·2H2O solution and continue stirring at room temperature for 12h. Let stand for 6h, centrifuge at 5000r / min for 10min, remove the solid, wash with ethanol 3-4 times, and vacuum dry to obtain Cu / Zn-MOF nanozymes.

[0032] 2. Characterization of Cu / Zn-MOF nanozymes

[0033] The prepared Cu / Zn-MOF nanozyme was analyzed by transmission electron microscopy (TEM). Figure 1 The results showed that the synthesized Cu / Zn-MOFs were aggregated spherical structures. X-ray diffraction analysis was used to evaluate the crystal structure of the Cu / Zn-MOFs, revealing multiple major diffraction peaks at 2θ angles of 14.1º, 20.0º, 24.5º, 31.8º, 34.9º, 43.2º, and 45.6º. Figure 2 The ), corresponding to the (110), (200), (211), (310), (222), (330), and (420) crystal planes, respectively; the distribution of characteristic functional groups on the Cu / Zn-MOF surface was determined by FT-IR. Figure 3 Several characteristic absorption peaks (such as 463 cm⁻¹, 611 cm⁻¹, and 764 cm⁻¹) are shown in the range of 450-780 cm⁻¹. These peaks are related to the stretching vibration of N-Zn. The broad absorption peak at 3405 cm⁻¹ is attributed to the stretching vibration of the OH bond. The absorption peak at 1632 cm⁻¹ corresponds to the stretching vibration of C=O. The strong absorption peaks at 1095 cm⁻¹, 1152 cm⁻¹, and 1208 cm⁻¹ correspond to the asymmetric and symmetric stretching vibrations of the CO bond. The absorption peaks at 1257 cm⁻¹ and 1443 cm⁻¹ correspond to the stretching vibrations of the CN bond, respectively. The absorption peak at 872 cm⁻¹ is attributed to the symmetric stretching vibration of the CH bond. The absorption peak at 1380 cm⁻¹ is attributed to the CC bond and is related to the in-plane tortuosity vibration of the aromatic ring. Figure 4In the high-resolution XPS spectrum of Cu 2p, the peaks of Cu 2p3 / 2 (932.5 eV) and Cu 2p1 / 2 (952.8 eV) show two satellite binding energy peaks at 942.3 and 962.0 eV, corresponding to the Cu 2p3 / 2 and Cu 2p1 / 2 energy levels, respectively, confirming the presence of Cu2+. The peak at 570.3 eV is attributed to Cu+, indicating the coexistence of Cu+ and Cu2+. Figure 4 In the high-resolution XPS spectrum of Zn 2p, two binding energy peaks are observed at 1021.8 and 1043.7 eV, corresponding to the Zn 2p3 / 2 and Zn 2p1 / 2 energy levels, respectively, confirming the presence of Zn2+. TEM, XPS, and FTIR data further support the successful synthesis of Cu / Zn-MOF.

[0034] 3. Evaluation of peroxidase activity of Cu / Zn-MOF nanozymes

[0035] 100 μL of 20 mmol / L o-phenylenediamine (OPD) was added to a 5 mL stoppered colorimetric tube, followed by 70 μL of 1 mg / mL Cu / Zn-MOF nanozyme solution and 160 μL of 50 mmol / L H2O2. The solution was diluted to 2.5 mL with 0.1 mol / L pH 6.5 MES buffer, vortexed to mix, and allowed to stand for 30 min. The fluorescence intensity was measured at excitation wavelengths of 421 nm and emission wavelengths of 564 nm. The results are as follows: Figure 5 Cu / Zn-MOF nanozymes exhibited strong peroxidase activity, while 5 μg / mL sodium dehydroacetate (DHA-S) significantly suppressed the fluorescence intensity of the system.

[0036] 4. Evaluation of the activity of Cu / Zn-MOF nanozymes laccase

[0037] Add 160 μL of 20 mmol / L 2,4-dichlorophenol (2,4-DP), 140 μL of 20 mmol / L 4-aminoantipyrine (4-AP), 50 μL of pH 6.5 0.1 mol / L LMES buffer, and 50 μL of 1 mg / mL Cu / Zn-MOF to a 5 mL stoppered colorimetric tube. Dilute the mixture to 1.5 mL with deionized water. After incubation for 30 min, measure the absorbance at 505 nm using a UV-Vis spectrophotometer. The results are as follows: Figure 6 Cu / Zn-MOF nanozymes exhibited strong laccase-like activity, while 5 μg / mL sodium dehydroacetate (DHA-S) significantly inhibited the absorbance of the system.

[0038] 5. Free radical capture test

[0039] This section uses different free radical scavengers to verify the presence of free radicals in the system. IPA (isopropanol), PBQ (p-benzoquinone), Na₂C₂O₄, Cr(VI), and tryptophan were used to monitor ·OH, ·O₂⁻, h⁺, e⁻, and 1O₂, respectively. Figure 7 As shown, the laccase-like activity of Cu / Zn-MOF significantly decreased after the addition of Na⁺ and Tryptophan, indicating that h⁺ and 1O₂ are the main active substances affecting the laccase-like activity of Cu / Zn-MOF; Figure 8 As shown, the fluorescence intensity of the system decreased significantly after the addition of PBQ and Tryptophan, indicating that ·O2- and 1O2 are the main active substances affecting the activity of Cu / Zn-MOF POD enzymes.

[0040] 6. Verification of Cu / Zn-MOF catalytic active sites

[0041] Since both natural laccase and laccase mimics use Cu as their catalytic active site, the active site of Cu / Zn-MOF is studied. This is based on the fact that thiocyanate (KSCN) is a chelating agent that blocks the catalytic active sites of transition metals such as Cu and Fe. Figure 9 As shown, the catalytic activity of Cu / Zn-MOF gradually decreased with increasing KSCN concentration, proving that Cu / Zn-MOF indeed uses Cu as the catalytic active center. Furthermore, the presence of oxygen vacancies (Ov) can enhance the catalytic activity of laccase; this study verified the presence of Ov by introducing the Ov-scavenging agent EDTA·2Na. Figure 10 As shown, with the gradual increase of EDTA·2Na concentration, the laccase-like catalytic activity of Cu / Zn-MOF decreased significantly, indicating that Cu / Zn-MOF has a large number of oxygen vacancies.

[0042] 7. Preparation of colorimetric working curve for dehydroacetic acid (DHA-S)

[0043] 160 μL of 20 mmol / L 2,4-DP, 140 μL of 20 mmol / L 4-AP, 50 μL of 1 mg / mL Cu / Zn-MOF, and DHA-S standard solutions of different concentrations (0.067–12 µg / mL) were mixed and diluted to 1.5 mL with 0.1 mol / L pH 6.5 MES buffer. After incubation for 30 min, the absorbance was measured at 505 nm to determine the linear relationship between DHA-S concentration and absorbance value. Figure 11 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are obtained and are shown in Table 1.

[0044] 8. Preparation of fluorescence working curve for dehydroacetic acid (DHA-S)

[0045] 100 μL of 20 mmol / L OPD, 70 μL of 1 mg / mL Cu / Zn-MOF nanozyme solution, 160 μL of 50 mmol / L H₂O₂, and DHA-S standard solutions of different concentrations (0.1-22 µg / mL) were mixed and diluted to 2.5 mL with 0.1 mol / L pH 6.5 MES buffer. After shaking and mixing, the mixture was allowed to stand for 30 min. The fluorescence intensity was measured at excitation wavelengths of 421 nm and emission wavelengths of 564 nm to determine the linear relationship between DHA-S concentration and fluorescence intensity. Figure 12 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are obtained and are shown in Table 1.

[0046]

[0047] 9. Method Specificity Study: The selectivity of the method was evaluated by replacing dehydroacetic acid (DHA-S) with other preservatives (sodium benzoate, potassium sorbate, sodium diacetate, sodium propionate, calcium propionate, methylparaben). Figure 13 (a, b) Potentially interfering substances (such as NH4+, Ca2+, Na+, Co2+, Zn2+, Mg2+, Mn2+, starch, sucrose, white sugar, sodium saccharin, yeast, potassium sorbate, sodium diacetate) were added to the system to evaluate the method's anti-interference ability. The effects of DHA-S and other substances on the Cu / Zn-MOF nanozyme activity and POD activity in step 4 were also investigated. Figure 14 (a, b)); where the concentration of DHA-S and other preservatives is 0.5 μg / mL, and the concentration of coexisting ions is 50 μg / mL. As can be seen from the figure, the Cu / Zn-MOF nanozyme detection system has good selectivity and specificity. DHA-S significantly inhibits the oxidation reaction, while other substances are almost absent. The method for determining DHA-S has good selectivity and specificity.

[0048] 10. Determination of DHA-S in food samples

[0049] (1) Sample preparation for DHA-S determination

[0050] Accurately weigh 1 g of the pulverized sample into a 15 mL centrifuge tube, add 1 mL NaOH (20 g / L) and 1 mL ZnSO4·7H2O (120 g / L), then bring the volume to 10 mL with deionized water. Place the tube in an ultrasonic cleaner and sonicate for 10 min, then let it stand for 30 min. Subsequently, centrifuge the mixture at 8000 rpm for 10 min, and use the supernatant as the sample assay solution.

[0051] (2) Sample determination: Under the same test conditions as the working curves in steps 7 and 8, the spiked recovery test in step (1) was carried out, and the results are shown in Table 2. The results show that the spiked recovery rate of DHA-S was between 92.1% and 106.4%, and the RSD was less than 5% (n=3). The results indicate that the method established in this invention can be used for the determination of DHA-S in food samples.

[0052]

[0053] Note: Colorimetric / Fluorescence

[0054] The DHA-S determination method established in this invention has advantages in practical testing because it involves fewer processing steps, shorter processing time, lower processing cost, simpler operation, and does not require large instruments or equipment.

Claims

1. A method for rapid detection of sodium dehydroacetate using a dual-mode colorimetric / fluorescence method with a peroxidase / laccase nanozyme, characterized in that, Includes the following steps: (1) Dissolve 0.080-0.100g CuCl2·2H2O in 10-15 mL methanol, 1.00-1.20g 1-hydroxybenzotriazole in 10-15 mL methanol, and 0.100-0.150g Zn(OAc)2·2H2O in 10-15 mL methanol. Sonicate each solution for 5-10 min to ensure complete dissolution. Slowly add the CuCl2·2H2O solution to the 1-hydroxybenzotriazole solution and stir for 5-10 min. Then slowly add the Zn(OAc)2·2H2O solution and continue stirring at room temperature for 12-15 h. Let stand for 5-10 h, centrifuge, remove the solid, wash with ethanol 3-4 times, and vacuum dry to obtain Cu / Zn-MOF nanozyme. (2) Add 2,4-dichlorophenol and 4-aminoantipyrine solution to pH 6.5 MES buffer solution, add Cu / Zn-MOF nanozyme and sodium dehydroacetate standard solution of different concentrations, mix well, incubate at room temperature for 20-30 min, measure absorbance at 505 nm wavelength, determine the linear relationship between sodium dehydroacetate concentration and absorbance value, and obtain regression equation; (3) Add o-phenylenediamine and H2O2 solution to pH 6.5 MES buffer solution, add Cu / Zn-MOF nanozyme and sodium dehydroacetate standard solution of different concentrations, mix well, incubate at room temperature for 20-30 min, and determine the linear relationship between sodium dehydroacetate concentration and fluorescence intensity by measuring the change in fluorescence intensity at 564 nm emission wavelength when the excitation wavelength is 421 nm, and obtain the regression equation. (4) The absorbance and fluorescence intensity of the sample solution to be tested are measured according to the methods in steps (2) and (3). The absorbance and fluorescence intensity are then substituted into the regression equation to obtain the concentration of sodium dehydroacetate in the sample solution to be tested.

2. The method for rapid detection of sodium dehydroacetate using a peroxidase / laccase nanoenzyme colorimetric / fluorescence dual-mode according to claim 1, characterized in that: The concentration of Cu / Zn-MOF nanozyme solution was 1 mg / mL, and the addition amount was 50-100 μL; the concentration of 2,4-dichlorophenol solution was 20 mmol / L, and the addition amount was 100-200 μL; the concentration of 4-aminoantipyrine solution was 20 mmol / L, and the addition amount was 100-200 μL; the concentration of o-phenylenediamine solution was 20 mmol / L, and the addition amount was 100-200 μL; the concentration of H2O2 solution was 50 mmol / L, and the addition amount was 100-200 μL.

3. The method for rapid detection of sodium dehydroacetate using a peroxidase / laccase nanoenzyme colorimetric / fluorescence dual-mode according to claim 1, characterized in that: In step (1), centrifugation is performed at 5000-10000 r / min for 5-10 min.

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