Method for rapidly detecting sodium dehydroacetate by nano-enzyme base

Abstract

According to the method, phosphomolybdic acid, copper chloride and ferric chloride serve as precursors, copper and iron-containing phosphomolybdic acid nano-enzyme (Cu, Fe-PMs) with peroxidase-like activity is synthesized through a microwave method, under the acidic condition, on one hand, the surface of Cu and Fe-PMs carries negative charges, high affinity is generated between Cu and Fe-PMs and a substrate TMB with positive charges, and on the other hand, the surface of Cu and Fe-PMs has negative charges; on the other hand, Cu / Fe / Mo three-metal redox couple accelerates the regeneration of Fe < 2 + > and plays an important role in the activity of peroxidase, and meanwhile, due to the introduction of sodium dehydroacetate, Cu and Fe in Cu and Fe-PMs and pyranoid ring and keto O in sodium dehydroacetate interact through coordinate bonds to cause electron interaction, so that the peroxidase-like activity of Cu and Fe-PMs is enhanced, and the peroxidase-like activity of Cu and Fe-PMs is improved. And the Fenton reaction is enhanced through transfer of electrons among the sodium dehydroacetate, the Cu and the Fe-PMs. When Cu and Fe-PMs oxidize TMB and H2O2, sodium dehydroacetate enhances the absorbance of the system, and on the basis, a novel colorimetric rapid detection method of sodium dehydroacetate is established.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a method for rapid detection of sodium dehydroacetate based on a nanoenzyme. Background Art

[0002] Dehydroacetic acid (DHA) has antimicrobial properties and is often used as a preservative in food production. With increasing attention to food safety, researchers are also concerned about the health risks of food additives. Studies have shown that DHA and its sodium salt (DHA-S) are classified as low toxic according to the WHO chemical toxicity classification system, posing a potential threat to human health. However, some food manufacturers use DHA-S beyond the specified range or in excessive quantities. Therefore, rapid and accurate qualitative and quantitative determination of DHA-S content is crucial. Currently, reported methods for DHA-S determination include high-performance liquid chromatography, gas chromatography, and electrochemiluminescence. Colorimetric methods hold a prominent position in detection technologies due to their simplicity, speed, and lack of the need for extensive instrumentation. However, colorimetric detection of DHA-S is virtually unreported.

[0003] Polyoxometalates (POMs) have attracted particular interest in catalysis, biomedicine, and materials science due to their electronic and chemical versatility and unlimited options for constructing diverse molecular architectures. The ability of POMs to undergo reversible multi-electron redox processes makes them suitable as enzyme mimics in bio- and chemical sensing systems. Summary of the Invention

[0004] The present invention provides a nanozyme-based method for rapid detection of sodium dehydroacetate. The method uses phosphomolybdic acid, copper chloride and ferric chloride as precursors, and synthesizes copper-iron phosphomolybdic acid nanozymes (Cu,Fe-PMs) with peroxidase-like activity by microwave method. Under acidic conditions, the Cu,Fe-PMs have a negative surface charge and generate a high affinity with the positively charged substrate TMB. On the other hand, the Cu / Fe / Mo trimetallic redox couple accelerates the Fe 2+ The regeneration of Cu,Fe-PMs plays a key role in peroxidase-like activity. The introduction of sodium dehydroacetate simultaneously enhances the peroxidase-like activity of Cu,Fe-PMs by causing electronic interactions between the Cu and Fe in the Cu,Fe-PMs and the pyran ring and keto group O in the sodium dehydroacetate through coordination bonds. This enhances the Fenton reaction through electron transfer between the sodium dehydroacetate and Cu,Fe-PMs. When Cu,Fe-PMs oxidize TMB and H₂O₂, sodium dehydroacetate enhances the absorbance of the system. Based on this, a new colorimetric rapid detection method for sodium dehydroacetate has been established. This method selectively detects sodium dehydroacetate, without interference from other substances. It boasts high sensitivity, strong specificity, simple operation, and rapidity.

[0005] The method for rapid detection of sodium dehydroacetate by nanoenzyme of the present invention is as follows: (1) Dissolve 0.25-0.30 g of phosphomolybdic acid, 0.30-0.35 g of copper chloride, 0.75-0.85 g of ferric chloride, and 0.20-0.30 g of dopamine in 50-70 mL of pure water, stir at room temperature for 30-40 min, heat at 140-160 °C in a microwave (500-1000 W) for 3-4 h, cool to room temperature, centrifuge, collect the precipitate, wash with anhydrous ethanol and pure water 3-4 times, respectively, and vacuum dry to obtain Cu,Fe-PMs nanozyme; (2) Add Cu,Fe-PMs nanozyme solution to sodium dehydroacetate standard solution of different concentrations, then add 3,3',5,5'-tetramethylbenzidine TMB solution and H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL, shake well to generate blue oxide oxTMB, let it stand for 10-15 min, measure the absorbance at a wavelength of 654 nm, establish a quantitative relationship between absorbance and sodium dehydroacetate concentration, draw a standard curve, and obtain the regression equation; (3) Extract sodium dehydroacetate from the sample to be tested to obtain a sample assay solution. Add Cu,Fe-PMs nanozyme solution to the sample assay solution, then add 3,3',5,5'-tetramethylbenzidine TMB solution and H2O2, add 0.1 mmol / L pH4.0 HAc-NaAc buffer to 3 mL, shake well to generate blue oxide oxTMB, let it stand for 10-15 min, measure the absorbance at a wavelength of 654 nm, and substitute the absorbance into the regression equation in step (2) to obtain the sodium dehydroacetate content in the sample.

[0006] The concentration of the Cu,Fe-PMs nanozyme solution is 1 mg / mL, and the addition amount is 50-100 μL; the concentration of TMB is 50 mmol / L, and the addition amount is 50-100 μL; the concentration of the H2O2 solution is 50 mmol / L, and the addition amount is 50-100 μL.

[0007] In the step (1), the centrifugation is carried out at 8000-10000 r / min for 5-10 min.

[0008] Advantages and technical effects of the present invention: 1. The present invention prepares copper-iron phosphomolybdate nanozymes (Cu,Fe-PMs). Under acidic conditions, the Cu,Fe-PMs have a negative surface charge and a high affinity with the positively charged substrate TMB. On the other hand, the Cu / Fe / Mo trimetallic redox couple accelerates the Fe 2+The regeneration of Cu,Fe-PMs plays a key role in peroxidase-like activity. The introduction of sodium dehydroacetate also enhances the peroxidase-like activity of Cu,Fe-PMs by causing electronic interactions between the Cu and Fe in the Cu,Fe-PMs and the pyran ring and keto group O in the sodium dehydroacetate through coordination bonds. This interaction enhances the Fenton reaction through electron transfer between the sodium dehydroacetate and Cu,Fe-PMs. When Cu,Fe-PMs oxidize TMB and H2O2, sodium dehydroacetate enhances the absorbance of the system. Based on this, a new colorimetric rapid detection method for sodium dehydroacetate was established.

[0009] 2. The colorimetric detection method for sodium dehydroacetate established in the present invention has a wide linear range, high detection sensitivity, and a detection limit of 0.5 μg / L. Coexisting substances do not interfere with the determination, and the method has good selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 TEM image of the Cu,Fe-PMs nanozyme prepared in Example 1 of the present invention; Figure 2 Zeta potential diagram of Cu, Fe-PMs and Cu, Fe-PMs+TMB in Example 1; Figure 3 The ultraviolet absorption spectra of TMB, Cu,Fe-PMs+TMB, and Cu,Fe-PMs+TMB+H2O2 in Example 1 of the present invention are shown; Figure 4 This is the fluorescence spectrum of •OH detected by terephthalic acid; Figure 5 •OH spectrum detected by electron spin resonance spectrometer; Figure 6 The linear regression equation and absorption spectrum of the Cu,Fe-PMs +TMB + H2O2 system for detecting DHA-S in Example 1; Figure 7 The results of the effects of other preservatives on the DHA-S detection system in Example 1 are as follows; Figure 8 This is the result of the influence of other coexisting ions on the DHA-S detection system in Example 1. DETAILED DESCRIPTION

[0011] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0012] Example 1: Determination of Sodium Dehydroacetate in Orange Juice Samples 1. Preparation of Cu,Fe-PMs nanozymes (1) Dissolve 0.25 g of phosphomolybdic acid, 0.30 g of copper chloride, 0.75 g of ferric chloride, and 0.20 g of dopamine in 50 mL of pure water. Stir at room temperature for 30 min, then heat in a microwave oven at 600 W and 140 °C for 4 h. Cool to room temperature, centrifuge at 10,000 rpm for 5 min, collect the precipitate, wash with anhydrous ethanol and pure water 3-4 times, and vacuum dry to obtain Cu,Fe-PMs nanozyme. Figure 1 To prepare the TEM images of Cu,Fe-PMs, it showed a uniform regular structure with a lattice of 0.25 nm and a graphite-like structure; Figure 2 is the Zeta potential of Cu,Fe-PMs and Cu,Fe-PMs+TMB. It can be seen that Cu,Fe-PMs exhibits a strong negative charge, and after adding TMB, the negative charge of the Zeta potential is partially neutralized.

[0013] 2. Evaluation of peroxidase activity of Cu,Fe-PMs nanozymes In a 5 mL stoppered colorimetric tube, 100 μL of 5 mmol / L TMB (3,3',5,5'-tetramethylbenzidine) was added, along with 100 μL of 1 mg / mL Cu,Fe-PMs nanozyme solution and 100 μL of 50 mmol / L H2O2. The tube was diluted to 3 mL with 0.1 mmol / L pH 4.0 HAc-NaAc buffer solution, and the mixture was shaken and allowed to stand for 10 min. The absorbance was measured at a wavelength of 654 nm. The results are shown in Figure 2. Figure 3 , Cu,Fe-PMs nanozymes exhibit strong peroxidase activity.

[0014] 3. Detection of hydroxyl radicals (•OH) Terephthalic acid (TA) was used as a probe to detect •OH. TA was oxidized to 2-hydroxyterephthalic acid (TAOH) in the presence of •OH. 100 μL of 1 mg / mL Cu,Fe-PMs nanozyme, 100 μL of 5 mg / mL TA, and 100 μL of 50 mmol / L H2O2 were added to 2.7 mL of pH 4.0 acetic acid-sodium acetate buffer solution, mixed thoroughly, and incubated at room temperature for 4 h. The fluorescence was measured at a wavelength of 425 nm under an excitation wavelength of 310 nm to explore its catalytic activity. The results are shown in Figure 2. Figure 4 , Cu,Fe-PMs+ H2O2 has a strong ability to generate •OH, and the •OH was determined by electron spin resonance spectrometer (EPR), and the results were consistent with the TA probe detection results ( Figure 5 ).

[0015] 4. Preparation of working curve of sodium dehydroacetate (DHA-S) In a 5 mL stoppered colorimetric tube, add 0.0017-2.5 μg / mL DHA-S, 100 μL of 50 mmol / L TMB solution, and 100 μL of 50 mmol / L H2O2. Add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL and shake well to generate blue oxidized oxTMB. Let it stand for 10 min and measure the absorbance at 654 nm. Draw a standard curve with DHA-S concentration as the horizontal axis and absorbance A as the vertical axis to obtain the regression equation, see [1]. Figure 6 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1.

[0016]

[0017] 5. Method specificity investigation: Replace sodium dehydroacetate (DHA-S) with other preservatives (benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, sodium propionate, sodium diacetate) and coexisting ions (Na + , K + , Ca 2+ Mg 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 、Cl - 、SO4 2- 、NO3 - ), detect the effects of DHA-S and other substances on Cu-PTs@PDA nanozymes in the detection system of step 4; the concentration of DHA-S and other preservatives was 0.5 μg / mL, and the concentration of coexisting ions was 50 μg / mL. The results are shown in Figure 7 、 8 ,As can be seen from the figure, the Cu,Fe-PMs nanoenzyme detection system has good ,selectivity. DHA-S significantly enhances the oxidation reaction, while ,other substances have almost no effect. ,This method has good selectivity for the determination of DHA-S.

[0018] 6. Determination of sodium dehydroacetate in orange juice samples (1) Sample processing for determination of DHA-S Weigh 2.5 g of sample (accurate to 0.001 g) and place it in a 50 mL centrifuge tube. Add approximately 10 mL of water and adjust the pH to 7.5 with 20 g / L sodium hydroxide solution. Transfer to a 50 mL volumetric flask, dilute to the mark with water, and shake well. Place in a centrifuge tube and centrifuge at 4000 rpm for 10 min. Take 20 mL of the supernatant and adjust the pH to 5 with 10% formic acid solution, and adjust the volume to 25 mL. Pass 5 mL of the supernatant through an activated C18 solid-phase extraction column. Rinse with 5 mL of water and elute with 2 mL of 70% methanol solution. Collect 2 mL of the eluate, vortex to mix, and filter through a 0.45 μm organic filter to obtain the sample solution. (2) Determination of DHA-S in orange juice samples: Add 100 μL of sample solution, 100 μL of 50 mmol / L TMB solution, and 100 μL of 50 mmol / L H2O2 to a 5 mL stoppered colorimetric tube. Add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL and shake well to generate blue oxidized oxTMB. Let it stand for 10 min and measure the absorbance at a wavelength of 654 nm. Substitute it into the regression equation in step 4. DHA-S was not detected.

[0019] Example 2: Determination of Sodium Dehydroacetate in Fermented Bean Curd Samples 1. Preparation of Cu,Fe-PMs nanozymes (1) Dissolve 0.30 g of phosphomolybdic acid, 0.35 g of copper chloride, 0.85 g of ferric chloride, and 0.30 g of dopamine in 70 mL of pure water. Stir at room temperature for 40 min, then heat in a microwave at 800 W and 160 °C for 3 h. Cool to room temperature, centrifuge at 8000 r / min for 10 min, collect the precipitate, wash with anhydrous ethanol and pure water 3-4 times, and vacuum dry to obtain Cu,Fe-PMs nanozyme.

[0020] 2. Preparation of sodium dehydroacetate (DHA-S) working curve: same as Example 1.

[0021] 3. Determination of sodium dehydroacetate in fermented bean curd samples (1) Sample processing for determination of DHA-S Homogenize the sample using a stainless steel high-speed homogenizer. Weigh 2.5 g of sample (accurate to 0.001 g) and place it in a 25 mL centrifuge tube. Add approximately 10 mL of water and 5 mL of 20 g / L zinc sulfate solution. Adjust the pH to 7.5 with 20 g / L sodium hydroxide solution. Transfer the sample to a 25 mL volumetric flask, dilute to the mark with water, and shake well. Place the sample in a 25 mL centrifuge tube, ultrasonically extract for 10 minutes, and centrifuge at 4000 rpm for 10 minutes. Pass the supernatant through a 0.45 μm organic filter to obtain the sample solution. (2) Determination of DHA-S in fermented bean curd samples: Add 100 μL of sample solution, 100 μL of 50 mmol / L TMB solution, and 100 μL of 50 mmol / L H2O2 to a 5 mL stoppered colorimetric tube. Add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL and shake well to generate blue oxidized oxTMB. Let it stand for 10 min and measure the absorbance at a wavelength of 654 nm. Substitute it into the regression equation in step 4. The DHA-S content is 15 μg / g.

[0022] Example 3: Determination of Sodium Dehydroacetate in Cake Samples 1. Preparation of Cu,Fe-PMs nanozymes (1) Dissolve 0.30 g of phosphomolybdic acid, 0.32 g of copper chloride, 0.80 g of ferric chloride, and 0.25 g of dopamine in 60 mL of pure water. Stir at room temperature for 35 min, then heat in a microwave at 1000 W and 150 °C for 4 h. Cool to room temperature, centrifuge, collect the precipitate, wash it with anhydrous ethanol and pure water 3-4 times, respectively, and vacuum dry to obtain Cu,Fe-PMs nanozyme.

[0023] 2. Preparation of sodium dehydroacetate (DHA-S) working curve: same as Example 1.

[0024] 3. Determination of sodium dehydroacetate in cake samples (1) Sample processing for determination of DHA-S Crush the cake using a grinder and mix thoroughly. Weigh 2 g of the sample (accurate to 0.001 g) and place it in a 25 mL centrifuge tube. Add 10 mL of water and 5 mL of a 20 g / L zinc sulfate solution. Adjust the pH to 7.5 with a 20 g / L sodium hydroxide solution. Transfer the sample to a 25 mL volumetric flask, dilute to the mark with water, and shake thoroughly. Place the sample in a centrifuge tube and ultrasonically extract for 10 minutes. Centrifuge at 4000 rpm for 10 minutes. Pass the supernatant through a 0.45 μm organic filter to obtain the sample solution. (2) Determination of DHA-S in cake samples: Add 50 μL of sample solution, 100 μL of 50 mmol / L TMB solution, and 100 μL of 50 mmol / L H2O2 to a 5 mL stoppered colorimetric tube. Add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL and shake well to generate blue oxidized oxTMB. Let it stand for 10 min and measure the absorbance at a wavelength of 654 nm. Substitute it into the regression equation in step 4. The DHA-S content is 205 μg / g.

[0025] (3) Recovery and precision experiments Three different concentrations of DHA-S standard solutions were added to the three samples respectively; each concentration was measured three times in parallel, and the spiked recovery was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 2; the spiked recovery of DHA-S was measured to be between 94.8% and 103.9%, and the RSD was between 1.90% and 4.09%. This method has good accuracy and precision.

[0026]

[0027] The DHA-S determination method established in the present invention has the advantages of fewer processing steps, shorter time, lower processing cost, simple operation, and no need for large-scale instruments and equipment, thus having great advantages in actual detection.

Claims

1. A method for rapid detection of sodium dehydroacetate based on nanoenzymes, characterized in that: The following steps are involved: (1) Dissolve 0.25-0.30 g phosphomolybdic acid, 0.30-0.35 g copper chloride, 0.75-0.85 g ferric chloride, and 0.20-0.30 g dopamine in 50-70 mL pure water, stir at room temperature for 30-40 min, heat in a microwave at 500-1000 W and 140-160 ° C for 3-4 h, cool to room temperature, centrifuge, collect the precipitate, wash with anhydrous ethanol and pure water for 3-4 times, and vacuum dry to obtain Cu,Fe-PMs nanozyme; (2) Add Cu,Fe-PMs nanozyme solution to sodium dehydroacetate standard solution of different concentrations, then add 3,3',5,5'-tetramethylbenzidine TMB solution and H2O2, add 0.1 mmol / L pH 4.0 HAc-NaAc buffer to 3 mL, shake well to generate blue oxide oxTMB, let stand for 10-15 min, measure the absorbance at 654 nm, establish the quantitative relationship between absorbance and sodium dehydroacetate concentration, draw the standard curve, and obtain the regression equation; (3) Extract sodium dehydroacetate from the sample to be tested to obtain a sample assay solution, add Cu,Fe-PMs nanozyme solution to the sample assay solution, then add 3,3',5,5'-tetramethylbenzidine TMB solution and H2O2, add 0.1 mmol / L pH4.0 HAc-NaAc buffer to 3 mL, shake well to generate blue oxide oxTMB, let stand for 10-15 min, measure the absorbance at a wavelength of 654 nm, substitute the absorbance into the regression equation in step (2) to obtain the sodium dehydroacetate content in the sample.

2. The method for rapid detection of sodium dehydroacetate by nanoenzyme according to claim 1, characterized in that: The concentration of Cu,Fe-PMs nanozyme solution is 1 mg / mL, and the amount added is 50-100 μL; the concentration of TMB is 50 mmol / L, and the amount added is 50-100 μL; the concentration of H2O2 solution is 50 mmol / L, and the amount added is 50-100 μL.

3. The method for rapid detection of sodium dehydroacetate by nanoenzyme according to claim 1, characterized in that: In step (1), the centrifugation is carried out at 8000-10000 r / min for 5-10 min.

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