A nanoenzyme-based method for rapid detection of sodium dehydroacetate

Abstract

The present invention discloses a method for rapid detection of sodium dehydroacetate based on nanozyme. This method uses phosphomolybdic acid, copper chloride and iron chloride as precursors, and synthesizes copper-iron-phosphomolybdic acid nanozyme (Cu,Fe-PMs) with peroxidase-like activity by microwave method. Under acidic conditions, on the one hand, the surface of Cu,Fe-PMs is negatively charged, which has a high affinity for the positively charged substrate TMB. On the other hand, the Cu / Fe / Mo trimetal redox couple accelerates the regeneration of Fe 2+ , which plays an important role in peroxidase-like activity. At the same time, the introduction of sodium dehydroacetate causes an electronic interaction through the coordination bond between Cu and Fe in Cu,Fe-PMs and the pyran ring and keto O in sodium dehydroacetate, thereby enhancing the peroxidase-like activity of Cu,Fe-PMs. The Fenton reaction is enhanced by the transfer of electrons between 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 is established.

Description

Technical Field

[0001] The 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 antibacterial properties and is often used as a preservative in food production. As people pay more attention to food safety, various researchers have also begun to pay attention to the harm of food additives to human health. Studies have shown that DHA and its sodium salt (DHA-S) belong to the low toxicity level in the WHO chemical toxicity classification standard, which poses a potential threat to human health, but some food production companies use DHA-S beyond the scope or in excess. Therefore, it is particularly important to quickly and accurately detect the content of DHA-S qualitatively and quantitatively. At present, the reported methods for determining DHA-S include: high performance liquid chromatography, gas chromatography, electrochemiluminescence, etc. The colorimetric method occupies an important position in detection technology because it is simple and fast to operate and does not require large instruments and equipment. The colorimetric detection of DHA-S has almost never been reported.

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

[0004] The present invention provides a method for rapid detection of sodium dehydroacetate based on nanozymes. 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 charge on the surface 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 an important role in the peroxidase-like activity. At the same time, the introduction of sodium dehydroacetate causes the Cu and Fe in Cu,Fe-PMs to interact with the pyran ring and keto group O in sodium dehydroacetate through coordination bonds, thereby enhancing the peroxidase-like activity of Cu,Fe-PMs and enhancing the Fenton reaction by transferring electrons between sodium dehydroacetate and Cu,Fe-PMs. When Cu,Fe-PMs oxidizes TMB and H2O2, sodium dehydroacetate enhances the absorbance of the system. Based on this, a new colorimetric rapid detection method for sodium dehydroacetate is established. The method of the present invention can only selectively detect sodium dehydroacetate, and other substances have no interference. The method has the characteristics of 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:

[0006] (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 at 140-160 °C for 3-4 h in a microwave (500-1000 W), 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;

[0007] (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;

[0008] (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.

[0009] The concentration of the 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 the H2O2 solution is 50 mmol / L, and the amount added is 50-100 μL.

[0010] In the step (1), the centrifugation is carried out at 8000-10000 r / min for 5-10 min.

[0011] Advantages and technical effects of the present invention:

[0012] 1. The present invention prepares copper-iron phosphomolybdic acid nanozymes (Cu,Fe-PMs). Under acidic conditions, the surface of Cu,Fe-PMs has a negative charge, which produces 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 an important role in the peroxidase-like activity. At the same time, the introduction of sodium dehydroacetate causes the Cu and Fe in Cu,Fe-PMs to interact with the pyran ring and keto group O in sodium dehydroacetate through coordination bonds, thereby enhancing the peroxidase-like activity of Cu,Fe-PMs and enhancing the Fenton reaction through the transfer of electrons between sodium dehydroacetate and Cu,Fe-PMs. When Cu,Fe-PMs oxidizes 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.

[0013] 2. The sodium dehydroacetate colorimetric detection method established by the present invention has a wide linear range, high detection sensitivity, and a detection limit of 0.5 μg / L. The coexisting substances do not interfere with the determination, and the method has good selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 TEM image of the Cu,Fe-PMs nanozyme prepared in Example 1 of the present invention;

[0015] Figure 2 Zeta potential diagram of Cu,Fe-PMs and Cu,Fe-PMs+TMB in Example 1;

[0016] Figure 3 The ultraviolet absorption spectra of TMB, Cu,Fe-PMs+TMB, Cu,Fe-PMs+TMB+H2O2 in Example 1 of the present invention are shown;

[0017] Figure 4 This is the fluorescence spectrum of •OH detected by terephthalic acid;

[0018] Figure 5 •OH spectrum detected by electron spin resonance spectrometer;

[0019] Figure 6 The linear regression equation and absorption spectrum of Cu,Fe-PMs +TMB + H2O2 system for detecting DHA-S in Example 1;

[0020] Figure 7 The results of the effects of other preservatives on the DHA-S detection system in Example 1;

[0021] Figure 8This is the result of the effect of other coexisting ions on the DHA-S detection system in Example 1. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0023] Example 1: Determination of Sodium Dehydroacetate in Orange Juice Samples

[0024] 1. Preparation of Cu,Fe-PMs nanozymes

[0025] (1) Dissolve 0.25 g phosphomolybdic acid, 0.30 g copper chloride, 0.75 g ferric chloride, and 0.20 g dopamine in 50 mL pure water, stir at room temperature for 30 min, heat in a microwave at 600 W and 140 °C for 4 h, cool to room temperature, centrifuge at 10,000 r / min for 5 min, collect the precipitate, wash with anhydrous ethanol and pure water for 3-4 times, and vacuum dry to obtain Cu,Fe-PMs nanozyme; Figure 1 To prepare the TEM images of Cu,Fe-PMs, it shows a uniform regular structure, and the lattice of 0.25 nm is 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 exhibit a strong negative charge, and after adding TMB, the negative charge of the Zeta potential is partially neutralized.

[0026] 2. Evaluation of peroxidase activity of Cu,Fe-PMs nanozymes

[0027] In a 5 mL stoppered colorimetric tube, add 100 μL of 5 mmol / L TMB (3,3',5,5'-tetramethylbenzidine), 100 μL of 1 mg / mL Cu,Fe-PMs nanozyme solution, 100 μL of 50 mmol / L H2O2, dilute to 3 mL with 0.1 mmol / L pH 4.0 HAc-NaAc buffer solution, shake and mix, let stand for 10 min, and measure the absorbance at a wavelength of 654 nm; the results are as follows: Figure 3 , Cu,Fe-PMs nanozymes exhibit strong peroxidase activity.

[0028] 3. Detection of hydroxyl radicals (•OH)

[0029] 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 425 nm under an excitation wavelength of 310 nm to explore its catalytic activity. The results are shown in Table 1. Figure 4 , Cu,Fe-PMs+ H2O2 has a strong ability to produce •OH. At the same time, the electron spin resonance spectrometer (EPR) was used to measure •OH, and the results were consistent with the TA probe detection results ( Figure 5 ).

[0030] 4. Preparation of working curve of sodium dehydroacetate (DHA-S)

[0031] 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, shake well to generate blue oxidized oxTMB, let 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 Figure 6 ; The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1.

[0032]

[0033] 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 effect of DHA-S and other substances on Cu-PTs@PDA nanozyme 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 obviously enhances the oxidation reaction, while other ,substances have almost no effect. The method has good ,selectivity for determining DHA-S.

[0034] 6. Determination of sodium dehydroacetate in orange juice samples

[0035] (1) Sample processing for determination of DHA-S

[0036] Weigh 2.5 g of the sample (accurate to 0.001 g), place it in a 50 mL centrifuge tube, add about 10 mL of water, 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 r / min for 10 min. Take 20 mL of the supernatant and adjust the pH to 5 with 10% formic acid solution, and make up to 25 mL. Take 5 mL and pass it through an activated C18 solid phase extraction column. Rinse with 5 mL of water, elute with 2 mL of 70% methanol solution, collect 2 mL of eluate, vortex mix, pass through a 0.45 μm organic filter membrane, and obtain the sample test solution;

[0037] (2) Determination of DHA-S in orange juice samples: In a 5 mL stoppered colorimetric tube, add 100 μL of sample solution, 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. Shake well to generate blue oxidized oxTMB. Let stand for 10 min. Measure the absorbance at a wavelength of 654 nm. Substitute into the regression equation in step 4. DHA-S was not detected.

[0038] Example 2: Determination of Sodium Dehydroacetate in Fermented Bean Curd Samples

[0039] 1. Preparation of Cu,Fe-PMs nanozymes

[0040] (1) Dissolve 0.30 g phosphomolybdic acid, 0.35 g copper chloride, 0.85 g ferric chloride, and 0.30 g dopamine in 70 mL pure water. Stir at room temperature for 40 min, heat in a microwave at 800 W and 160 °C for 3 h, cool to room temperature, and centrifuge at 8000 r / min for 10 min. Collect the precipitate, wash it with anhydrous ethanol and pure water for 3-4 times, and dry it in vacuum to obtain Cu,Fe-PMs nanozyme.

[0041] 2. Preparation of working curve of sodium dehydroacetate (DHA-S): same as in Example 1.

[0042] 3. Determination of sodium dehydroacetate in fermented bean curd samples

[0043] (1) Sample processing for determination of DHA-S

[0044] The sample was homogenized with a stainless steel high-speed homogenizer, 2.5 g of the sample (accurate to 0.001 g) was weighed and placed in a 25 mL centrifuge tube, about 10 mL of water and 5 mL of 20 g / L zinc sulfate solution were added, the pH was adjusted to 7.5 with 20 g / L sodium hydroxide solution, transferred to a 25 mL volumetric flask, diluted to the mark with water, and shaken. Placed in a 25 mL centrifuge tube, ultrasonically extracted for 10 min, centrifuged at 4000 r / min for 10 min, the supernatant was filtered through a 0.45 μm organic filter membrane to obtain the sample test solution;

[0045] (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. Shake well to generate blue oxidized oxTMB. Let stand for 10 min. Measure the absorbance at a wavelength of 654 nm. Substitute into the regression equation in step 4. The DHA-S content is 15 μg / g.

[0046] Example 3: Determination of Sodium Dehydroacetate in Cake Samples

[0047] 1. Preparation of Cu,Fe-PMs nanozymes

[0048] (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, 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 for 3-4 times, respectively, and vacuum dry to obtain Cu,Fe-PMs nanozyme.

[0049] 2. Preparation of working curve of sodium dehydroacetate (DHA-S): same as in Example 1.

[0050] 3. Determination of sodium dehydroacetate in cake samples

[0051] (1) Sample processing for determination of DHA-S

[0052] Use a grinder to crush the cake, mix well, weigh 2 g of the sample (accurate to 0.001 g), place it in a 25 mL centrifuge tube, add 10 mL of water, 5 mL of 20 g / L zinc sulfate solution, adjust the pH to 7.5 with 20 g / L sodium hydroxide solution, transfer to a 25 mL volumetric flask, dilute to the scale with water, and shake well. Place in a centrifuge tube, ultrasonically extract for 10 min, centrifuge at 4000 r / min for 10 min, take the supernatant and filter it through a 0.45 μm organic filter membrane to obtain the sample test solution;

[0053] (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. Shake well to generate blue oxidized oxTMB. Let stand for 10 min. Measure the absorbance at a wavelength of 654 nm. Substitute into the regression equation in step 4. The DHA-S content is 205 μg / g.

[0054] (3) Recovery and precision experiments

[0055] Three different concentrations of DHA-S standard solutions were added to three samples respectively; each concentration was measured three times in parallel, the spiked recovery rate was calculated, and the relative standard deviation (RSD) was calculated. The results are shown in Table 2; the spiked recovery rate 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.

[0056]

[0057] The DHA-S determination method established in the present invention has the advantages of fewer processing steps, shorter time, lower processing cost, easier 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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