Method for detecting content of ferric ammonium citrate in table salt by using nitrogen-doped carbon dot nano-enzyme and application

Abstract

The invention relates to the technical field of chemical analysis and detection, and aims to provide a high-sensitivity and strong-specificity ferric ammonium citrate detection method aiming at the defect of determining the content of ferric ammonium citrate in a high-salt system so as to improve the detection precision of the content of ferric ammonium citrate in table salt. N-doped carbon dot nano-enzyme (N-CDs) is synthesized from octanamine, citric acid and ethidene diamine through a one-step microwave method, the N-CDs has peroxidase-like activity, can catalytically oxidize a substrate ABTS and H2O2 to generate a blue-green oxidation product (ox-ABTS) under a neutral condition and generate maximum absorption at the wavelength of 416 nm, ammonium ferric citrate can enhance the peroxidase-like activity of the N-CDs, and the N-CDs can catalytically oxidize the substrate ABTS and H2O2 to generate a blue-green oxidation product (ox-ABTS) and generate maximum absorption at the wavelength of 416 nm. The concentration of the ferric ammonium citrate in the sample can be measured according to the linear relationship between the concentration of the ferric ammonium citrate and the increase of the absorbance of the ox-ABTS, so that the green color of the ox-ABTS is deepened, and the concentration of the ferric ammonium citrate in the sample can be measured according to the linear relationship. Moreover, the N-CDs nano enzyme is good in water solubility and high in salt tolerance, and various ions in the salt hardly influence a detection result, so that the method also has good selection specificity and wider applicability.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a method and application of detecting the content of ammonium ferric citrate in table salt by utilizing nitrogen-doped carbon dot nanoenzymes. Background Art

[0002] Ammonium ferric citrate is a mixture of ferric citrate and ammonium citrate, but the content of ferric citrate and ammonium citrate in the product is not fixed and will vary with changes in synthesis conditions. According to GB 2760-2024, "National Food Safety Standard for the Use of Food Additives," ammonium ferric citrate can be added to salt and salt substitutes as an anti-caking agent to prevent salt products from agglomerating during storage and use, which affects the use of the product. The amount added should not exceed 25 mg / kg. Ammonium ferric citrate can also be added to food as a nutritional enhancer to increase the nutritional content of iron in food. It is widely used in the production of green food edible salt. However, if the content of ammonium ferric citrate is too high, it may cause irritation to the human gastric mucosa, resulting in adverse symptoms such as nausea, vomiting, and stomach pain. Therefore, testing ammonium ferric citrate is an important means to ensure the safety of salt products.

[0003] According to the provisions of QB / T 5776 light industry standard "Determination of anti-caking agent ammonium ferric citrate in edible salt", the conventional detection method for ammonium ferric citrate in edible salt is the spectrophotometric method, which uses a known iron standard solution (ferric sulfate) to prepare solutions of different concentrations, and measures its absorbance by a spectrophotometer. A standard curve is made based on the positive correlation between absorbance and iron ion content, and then the absorbance of the iron ion in the unknown solution is measured. The iron ion content is checked on the standard curve based on the absorbance, and finally the content of ammonium ferric citrate is calculated based on the iron ion content. However, the entire detection process of this method is in a high salt system, and ferric sulfate will coexist with the Na + , K + Mg 2+ , Ca 2+ 、Cl - , I - IO3 - 、SO4 2- Side reactions may occur, and even insoluble matter may be produced, resulting in a low detection content of ammonium ferric citrate and a lower detection accuracy.

[0004] Therefore, in view of the defects of the existing technology in determining the content of ammonium ferric citrate in a high salt system, it is necessary to propose a new method for detecting the content of ammonium ferric citrate in table salt, so as to improve the detection accuracy of the content of ammonium ferric citrate in table salt and provide a new detection idea for the setting of industry standards.

[0005] The above content is only used to assist in understanding the technical solution of the present invention and does not constitute an admission that the above content is prior art. Summary of the Invention

[0006] The main purpose of the present invention is to provide a method and application for detecting the content of ammonium ferric citrate (FAC) in table salt using nitrogen-doped carbon dot nanozymes (N-CDs). It aims to address the influence of coexisting ions in high-salt systems on the determination of ammonium ferric citrate content, and proposes a method and application for detecting the content of ammonium ferric citrate in table salt using nitrogen-doped carbon dot nanozymes, so as to improve the detection accuracy of the content of ammonium ferric citrate in table salt and provide a new detection idea for the setting of industry standards.

[0007] To achieve the above objectives, on the one hand, the present invention provides a method for detecting the content of ammonium ferric citrate in salt using nitrogen-doped carbon dot nanozymes, the method comprising the following steps:

[0008] S1, prepare standard solution

[0009] Preparation of ammonium ferric citrate standard sample solution: within the concentration range of 0-6 μg / mL, take nine groups of ammonium ferric citrate standard solutions with different concentrations according to an arbitrary concentration gradient and add them to nine colorimetric tubes respectively; then add nitrogen-doped carbon dot nanozyme solution, 2,2'-hydrazino-bis-3-ethylbenzothiazoline-6-sulfonic acid and H2O2 to the colorimetric tubes respectively, dilute to 4 mL with pH 7.0 phosphate buffer solution, mix well, and let stand for 5-10 minutes to obtain nine groups of different standard sample solutions.

[0010] Blank standard solution: Add nitrogen-doped carbon dot nanozyme solution, 2,2'-hydrazino-bis-3-ethylbenzothiazoline-6-sulfonic acid, and H2O2 to a colorimetric tube, dilute to 4 mL with pH 7.0 phosphate buffer solution, mix well, and let stand for 5-10 minutes to obtain a blank standard sample solution.

[0011] S2, prepare standard curve

[0012] Determine the absorbance of the standard solution at the maximum absorption wavelength, use the concentration of the ammonium ferric citrate standard solution as the horizontal axis and the corresponding absorbance difference ΔA as the vertical axis, ΔA=A-A0, draw a standard curve, and calculate the standard curve regression equation.

[0013] A is the absorbance of the ammonium ferric citrate standard sample solution at the maximum absorption wavelength;

[0014] A0 is the absorbance of the blank standard solution at the maximum absorption wavelength.

[0015] S3, detection of the content of ammonium ferric citrate in salt samples

[0016] Prepare salt sample solution: prepare salt sample solution with a concentration of 0.2 g / mL;

[0017] Take 0.5 mL of the salt sample solution, then add nitrogen-doped carbon dot nanozyme solution, 2,2'-hydrazino-bis-3-ethylbenzothiazoline-6-sulfonic acid and H2O2 into a colorimetric tube, dilute to 4 mL with pH 7.0 phosphate buffer solution, mix well, let stand for 5 to 10 minutes, measure the absorbance at the maximum absorption wavelength, and calculate the content of ammonium ferric citrate in the sample using the standard curve regression equation.

[0018] Optionally, the concentration of the nitrogen-doped carbon dot nanozyme solution is 1 mg / mL, and the added amount is 50 to 100 μL; the concentration of the 2,2'-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid is 5 mmol / L, and the added amount is 50 to 100 μL; the concentration of the H2O2 is 50 mmol / L, and the added amount is 50 to 100 μL.

[0019] Optionally, the amount of the nitrogen-doped carbon dot nanozyme solution added is 100 μL; the amount of the 2,2'-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid added is 100 μL; and the amount of H2O2 added is 100 μL.

[0020] Optionally, the maximum absorption wavelength is 416 nm.

[0021] On the other hand, the present application also proposes a method for preparing the nitrogen-doped carbon dot nanozyme solution as described above, the preparation method comprising the following steps:

[0022] Dissolve 20-30 mg of octopamine and 2.0-2.5 g of citric acid in 30-40 mL of deionized water, then add 50-100 μL of ethylenediamine, and ultrasonically treat for 20-30 min to obtain a mixed reagent; then perform microwave digestion on the mixed reagent, cool to room temperature after the reaction is completed to obtain a suspension; filter the suspension and then centrifuge to obtain a supernatant, and dry the supernatant to obtain nitrogen-doped carbon dot nanozyme; finally, add deionized water to obtain a nitrogen-doped carbon dot nanozyme solution.

[0023] Optionally, the specific operation of the microwave digestion is: the mixed reagent is subjected to 850W microwave to raise the reaction temperature to 180°C within 1 minute, and the reaction is continued for 1 to 2 hours.

[0024] Optionally, the specific operation of the filtration is to filter the suspension using a filter membrane with a pore size of 22 μm.

[0025] Optionally, the centrifugal speed is 8000-10000 r / min, and the time is 5-10 min.

[0026] Principle of the invention:

[0027] In high salt systems, nitrogen-doped carbon dot nanozymes (N-CDs) have high water solubility and salt tolerance. + , K + Mg 2+ , Ca 2+ 、Cl - , I - IO3 - 、SO4 2- No insoluble matter appears during the action, so when nitrogen-doped carbon dot nanozymes are used to detect the content of ammonium ferric citrate in table salt, the impact on the detection results of the content of ammonium ferric citrate is small and the detection accuracy is higher.

[0028] N-CDs not only have enzymatic catalytic activity, but also have photothermal effect, stable fluorescence and fluorescence conversion properties. Therefore, N-CDs can be used as a new type of fluorescent marker and can also be used in biological imaging.

[0029] Basic principles for making linear curves (UV spectrophotometry):

[0030] In the present invention, N-CDs has peroxidase-like activity and can act as a catalyst to cause an oxidation reaction between 2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and H2O2 under neutral conditions to generate a blue-green oxidation product (ox-ABTS). This color-developed oxidation product (ox-ABTS) has a maximum absorption at a wavelength of 416 nm and can be quantitatively analyzed by ultraviolet spectrophotometry. The addition of ammonium ferric citrate can enhance the peroxidase-like activity of N-CDs, causing the blue-green oxidation product (ox-ABTS) produced by the N-CDs-catalyzed oxidation of ABTS and H2O2 to have a deeper green color and a higher absorbance. Moreover, the concentration of the ammonium ferric citrate standard solution is linearly related to the increase in the absorbance of ox-ABTS. Therefore, by measuring the absorbance of ammonium ferric citrate standard solutions of different concentrations at the maximum absorption wavelength of 416 nm, a standard working curve can be drawn based on the quantitative relationship between the ammonium ferric citrate concentration and the absorbance, and a standard curve regression equation can be derived. Then the absorbance of the sample at 416 nm is measured, and the concentration of ammonium ferric citrate in the sample can be calculated through the regression equation, thereby achieving quantitative analysis of the content of ammonium ferric citrate in the salt sample.

[0031] The embodiment of the present invention provides a method for detecting the content of ammonium ferric citrate in salt using nitrogen-doped carbon dot nanozymes, which has at least the following beneficial effects:

[0032] 1. The present invention utilizes the peroxidase-like activity of N-CDs. Under neutral conditions, it can catalyze the oxidation of ABTS and react with H2O2 to generate a blue-green oxidation product (ox-ABTS) with a maximum absorption at 416nm. The addition of ammonium ferric citrate can enhance the peroxidase-like activity of N-CDs, causing the green color of ox-ABTS to deepen and the absorbance to increase. Therefore, based on the principle that the concentration of ammonium ferric citrate is linearly related to the absorbance of ox-ABTS, a new method for detecting the content of ammonium ferric citrate in a sample has been established. The method has the characteristics of high sensitivity, simple operation, and rapidity.

[0033] 2. In a high-salt system, the N-CDs prepared by the present invention have good water solubility and salt tolerance. No insoluble matter appears when reacting with the substrate ABTS. When detecting the content of ammonium ferric citrate in table salt, the measurement results are accurate, the spiked recovery rate reaches 96.95% to 103.88%, and the detection accuracy is high.

[0034] 3. The specificity test was used to test the standard sample solution, which can show that the Na + , K + Mg 2+ , Ca 2+ 、Cl - , I - IO3 - 、SO4 2- There is almost no impact on the detection results of the present invention, so the detection method of the present invention has very good selection specificity and wider applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the transmission electron microscopy TEM image of N-CDs nanozyme in Example 1.

[0036] Figure 2 The UV-vis absorption spectra of the peroxidase chromogenic substrates ABTS and H2O2 catalyzed by N-CDs and N-CDs+FAC in Example 1.

[0037] Figure 3 (a, b) are the Michaelis-Menten plots of ABTS and H2O2 fitted by N-CDs catalysis in Example 1 of the present invention.

[0038] Figure 4 (a, b) are the Michaelis-Menten plots of ABTS and H2O2 fitted by N-CDs+FAC in Example 1 of the present invention.

[0039] Figure 5 Fluorescence spectrum of ·OH detected by TA.

[0040] Figure 6 EPR detection OH spectrum.

[0041] Figure 7 This is the linear fitting curve for detecting FAC in the range of 0.05 to 5.81 μg / mL using N-CDs in Example 1 of the present invention.

[0042] Figure 8 N-CDs detected FAC selectivity results.

[0043] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0044] To better understand the above technical solutions, exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0045] Preparation principle of nitrogen-doped carbon dot nanozymes (N-CDs):

[0046] Carbon quantum dots (CDs) are a new type of quasi-spherical carbon nanomaterial with advantages such as small particle size, low toxicity, good biocompatibility and water solubility, and adjustable surface oxygen groups (hydroxyl, carbonyl, and carboxyl). Furthermore, CDs, as strong electron donors, can serve as novel reducing agents to promote the nucleation of metal nanoparticles and stabilize metal nanoparticles by preventing their aggregation in pure aqueous solutions.

[0047] To improve the properties of carbon nanomaterials, chemical doping can be used to modify their chemical and electronic properties. One approach involves adsorbing gases, metals, or organic molecules onto the material's surface. Another involves introducing heteroatoms, typically boron (B) or nitrogen (N) atoms, into the material's structure. Because B has one fewer outer electron than C, replacing C with B creates a hole. Conversely, replacing a C atom with N creates an extra electron. Nitrogen doped into CDs can inject electrons into the CDs, altering their original electronic structure. This strengthens bonding with ions and significantly increases the electron storage capacity of N-CDs, thereby altering the electronic and transport properties of the material. Nitrogen can effectively alter the luminescence properties of CDs and enhance the photoluminescence quantum yield (PLQY). Because N is similar in size to C and has five valence electrons, it can form covalent bonds with C atoms. Furthermore, the higher electron affinity and electronegativity of N enhance the chemical reactivity of CDs. Introducing N into CDs (N-CDs) can enhance various material properties. N-CDs exhibit excellent oxidase mimetic activity under light, which is induced by superoxide radicals (·O2- )-mediated, with a higher Vmax (1.59 μmol / s) and a lower Km (0.421 mmol / L).

[0048] Principle of Michaelis-Menten catalytic kinetic parameters determination:

[0049] With other parameters unchanged, steady-state kinetic analysis of the catalytic activity of FAC at different concentrations of ABTS and H2O2 was performed. The Michaelis-Menten kinetic equation was obtained by plotting the reciprocal of the reaction rate (V) against the reciprocal of the substrate concentration [S].

[0050] 1 / V=(Km / Vmax)(1 / [S])+1 / Km

[0051] V is the reaction rate / (mol / (L·s));

[0052] Km is the Michaelis constant / (mmol / L);

[0053] Vmax is the maximum reaction rate / (mol / (L·s));

[0054] [S] is the concentration of substrate H2O2 / (mmol / L).

[0055] The catalytic reaction increased significantly with the increase of H2O2 substrate concentration. N-CDs first reacted with H2O2 to produce hydroxyl radicals, which further oxidized the substrate ABTS to produce green ox-ABTS.

[0056] The reason for detecting hydroxyl radicals (·OH) is that ·OH reacts with benzoic acid to form hydroxybenzoic acid, which emits fluorescence at an excitation wavelength of 330 nm. Under acidic conditions, N-CDs catalyze H2O2 to produce hydroxyl radicals, which further oxidize the substrate. Detecting ·OH can be used to investigate the catalytic activity of N-CDs.

[0057] Based on the above description, an embodiment of a method and application of the present invention for detecting the content of ammonium ferric citrate in table salt using nitrogen-doped carbon dot nanozymes is proposed.

[0058] First embodiment

[0059] 1. Preparation of nitrogen-doped carbon dot nanozyme (N-CDs) solution: Dissolve 20-30 mg of octopamine and 2.0-2.5 g of citric acid in 30-40 mL of deionized water, add 50-100 μL of ethylenediamine, and sonicate for 20-30 minutes. Transfer the mixed solution to a polytetrafluoroethylene autoclave and place it in an 850W microwave digester for microwave digestion. Raise the reaction temperature to 180°C within 1 minute and continue the reaction for 1-2 hours. After the reaction is completed, cool naturally to room temperature, remove large particles with a 0.22 μm filter, and then centrifuge at high speed. The supernatant is vacuum-dried to obtain the nitrogen-doped carbon dot nanozyme. Finally, add deionized water to obtain the nitrogen-doped carbon dot nanozyme solution. The above nitrogen-doped carbon dot nanozyme can be prepared into a nitrogen-doped carbon dot nanozyme solution of a specific concentration according to the needs of subsequent experiments.

[0060] 2. The obtained nitrogen-doped carbon dot nanozymes were subjected to transmission electron microscopy (TEM) analysis. The results showed that the nitrogen-doped carbon dot nanozymes were spherical and well dispersed. The average particle size obtained from the illustration of the TEM particle size statistics was 2.85±0.72nm. High-resolution TEM (HRTEM) observed that the lattice spacing of the nitrogen-doped carbon dot nanozymes was 0.22nm, corresponding to the (100) crystal plane of graphitic carbon.

[0061] 3. Determination of N-CDs peroxidase activity: Add 100 μL of 1 mg / mL N-CDs or 100 μL of 1 μg / mL FAC + 100 μL of 1 mg / mL N-CDs, 100 μL of 5 mmol / L ABTS, and 100 μL of 50 mmol / L H2O2 to 2.7 mL of pH 7.0 phosphate buffer solution, mix thoroughly, incubate at room temperature for 10 minutes, and measure the absorbance at 416 nm using a UV-visible spectrophotometer. Measure each sample three times and take the average value. The results are as follows: Figure 2 ; As can be seen from the figure, N-CDs exhibits high peroxidase activity, and the addition of FAC enhances the enzyme activity of N-CDs.

[0062] 4. The experiment also carried out the determination of Michaelis-Menten catalytic kinetic parameters ( Figure 3 a, b, 4a, b and Table 1), the Michaelis constant K of N-CDs for substrates ABTS and H2O2 m The reaction rates were 4.038 mmol / L and 1.210 mmol / L, respectively, and the reaction rate constants were 8.724 × 10 -8 and 0.568×10 -8 mol / L·s, K of ABTS and H2O2 after adding FAC m are 1.311mmol / L and 0.521mmol / L, and the reaction rate constants are 31.78×10 -8 and 4.743×10-8 mol / L·s, indicating that the addition of FAC greatly enhanced the affinity and reaction rate of N-CDs with substrates ABTS and H2O2.

[0063] Table 1 Michaelis-Menten catalytic kinetic parameters

[0064]

[0065] 5. Detection of hydroxyl radical (·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 nanozyme N-CDs, 100 μL of 5 mg / mL TA, and 100 μL of 50 μmol / L H2O2 were added to 2.7 mL of pH 7.0 phosphate buffer solution, mixed thoroughly, and reacted at room temperature for 4 h. The fluorescence was measured at a wavelength of 410 nm under an excitation wavelength of 330 nm to explore its catalytic activity. The results are shown in Table 1. Figure 5 , N-CDs+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 6 ).

[0066] Second embodiment

[0067] The nitrogen-doped carbon dot nanozyme solution prepared in the first embodiment was used to determine the amount of ammonium ferric citrate in table salt. The concentration of the nitrogen-doped carbon dot nanozyme solution in this embodiment was 1 mg / mL. The specific detection steps included:

[0068] 1. Preparation of ferric ammonium citrate (FAC) working curve:

[0069] Ammonium ferric citrate standard solution: Within the concentration range of 0-6 μg / mL, take 100 μL of nine sets of ammonium ferric citrate standard solutions of different concentrations according to any concentration gradient and add them to nine colorimetric tubes respectively; then, add 100 μL of 1 mg / mL N-CDs, 100 μL of 5 mmol / L ABTS, and 100 μL of 50 mmol / L H2O2 to the colorimetric tubes respectively, dilute to 4 mL with pH 7.0 phosphate buffer solution, mix well, and let stand for 10 minutes to obtain nine sets of different standard sample solutions;

[0070] Blank standard sample solution: Add 100 μL of 1 mg / mL N-CDs, 100 μL of 5 mmol / L ABTS, and 100 μL of 50 mmol / L H2O2 to a colorimetric tube, dilute to 4 mL with pH 7.0 phosphate buffer solution, mix well, and let stand for 10 minutes to obtain a blank standard sample solution;

[0071] Measure the ultraviolet absorption curves of nine different standard sample solutions at 400-700 nm, record the absorbance at 416 nm (determine the maximum wavelength, and subsequent absorbance measurements are performed at this wavelength) (ΔA = A-A0, A0 is the absorbance of the blank standard sample solution, and A is the absorbance after adding ammonium ferric citrate solution), and draw a calibration curve (standard curve) as a function of the absorbance difference and FAC concentration, see Figure 7 b, and the obtained linear equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 2.

[0072] Table 2 Linear equation, correlation coefficient, relative standard deviation (RSD), linear range

[0073]

[0074] Limit of Detection (LOD): The lowest amount of analyte that can be detected in a sample. Linear range: 0.05–5.81 μg / mL, applicable to all ammonium ferric citrate standard solutions with concentrations between 0 and 6 μg / mL. "c" in the linear equation is the concentration of ammonium ferric citrate detected.

[0075] 2. Specificity investigation: Figure 8 Na coexisting in salt + , K + Mg 2+ , Ca 2+ 、Cl - , I - IO3 - 、SO4 2- The FAC concentration was 5 mg / kg, and the concentrations of all interfering substances were 100 times that of FAC. The results showed that only FAC had a significant enhancing effect, while the other substances had almost no effect, indicating that the method has good selectivity.

[0076] 3. Determination of ammonium ferric citrate in table salt

[0077] (1) Preparation of sample solution: Accurately weigh 10 g (accurate to 0.001 g) of sample, dissolve it in deionized water, and dilute to a 50 mL volumetric flask to prepare the sample solution;

[0078] (2) Sample determination: Accurately pipette 0.5 mL of the sample solution into a colorimetric tube, and simultaneously add 100 μL of 1 mg / mL N-CDs, 100 μL of 5 mmol / L ABTS, and 100 μL of 50 mmol / L H2O2. Dose the solution to 4 mL with pH 7.0 phosphate buffer solution, mix well, and let stand for 10 min. Measure the absorbance at a wavelength of 416 nm and substitute it into the regression equation of the standard working curve to calculate the results. The results are shown in Table 3. At the same time, a spike recovery test was performed, which was between 96.95% and 103.88%, with an RSD of less than 3% (n = 6).

[0079] At the same time, the sample was determined by the method of the reference document (Wei Feng et al., High Performance Liquid Chromatographic Detection Method of Ammonium Ferric Citrate in Table Salt, Chinese Condiments, 2017, 24(2)). The results are shown in Table 3. The content in "()" in "Detection Amount" is the determination result of this method. The results show that the method established by the present invention can be used for the determination of ammonium ferric citrate in table salt.

[0080] Table 3 Determination of low sodium salt samples and spiked recovery rates (n=6)

[0081]

[0082] After analyzing the data in Table 3, it can be shown that when the salt sample is low-sodium salt, the determination result is accurate, the spike recovery rate reaches 96.95% to 101.77%, the detection accuracy is high, the determination time is 10 minutes, and the detection efficiency is fast.

[0083] Third embodiment

[0084] In this embodiment, only the type of salt sample to be tested is changed, and seaweed iodized salt is selected for the test. The remaining steps are exactly the same as those in Example 2.

[0085] Table 4 Determination of seaweed iodine salt samples and spiked recovery rates (n=6)

[0086]

[0087] After analyzing the data in Table 4, it can be shown that when the salt sample is seaweed iodized salt, the determination result is accurate, the spiked recovery rate reaches 97.16% to 103.88%, the detection accuracy is high, the determination time is 9 minutes, and the detection efficiency is fast.

[0088] Fourth embodiment

[0089] In this embodiment, only the type of salt sample to be tested is changed, and non-iodized salt is selected for the test. The remaining steps are exactly the same as those in Example 2.

[0090] Table 5 The present invention measures the non-iodized salt sample and the recovery rate of spiked salt (n=6)

[0091]

[0092]

[0093] After analyzing the data in Table 5, it can be shown that when the salt sample is non-iodized salt, the determination result is accurate, the spiked recovery rate reaches 100.09% to 103.03%, the detection accuracy is high, the determination time is 10 minutes, and the detection efficiency is fast.

[0094] The first comparison

[0095] The test was conducted using low sodium salt in the same manner as in Example 2, but the determination method was the o-phenanthroline spectrophotometric method specified in the light industry standard "Determination of the Anticaking Agent Ammonium Ferric Citrate in Edible Salt" (QB / T 5776). The test steps were carried out in accordance with the test steps specified in the standard. The test results are as follows:

[0096] Table 6 Spectrophotometric determination of low sodium salt samples by o-phenanthroline and recovery of spiked samples (n=6)

[0097]

[0098] Under normal circumstances, the closer the spiked recovery is to 100%, the higher the data credibility is, and the higher the accuracy of detection is. After analyzing the data in Table 6, it can be known that, compared with the inventive method, the deviation value of the spiked recovery is larger when the method of industry standard is adopted to detect low sodium salt samples. That is, the accuracy of its detection result is lower than the accuracy of the inventive method when the method of industry standard is adopted. Simultaneously, the determination time is 30 minutes, which is longer than the determination time of the present invention.

[0099] Therefore, after comparison, it can be concluded that compared with the conventional standard detection method, the present invention has higher accuracy and shorter detection time.

[0100] The second comparison

[0101] The same test as in Example 3 was conducted using seaweed iodized salt, but the determination method was the o-phenanthroline spectrophotometric method specified in the light industry standard "Determination of the Anticaking Agent Ammonium Ferric Citrate in Edible Salt" (QB / T 5776). The test steps were carried out in accordance with the test steps specified in the standard. The test results are as follows:

[0102] Table 7 O-phenanthroline spectrophotometric determination of seaweed iodized salt samples and spiked recovery (n = 6)

[0103]

[0104] Analysis of the data in Table 7 reveals that the deviation in the spiked recovery rate for testing seaweed iodized salt samples using the industry standard method is greater than that of the present invention. This means that the accuracy of the test results using the industry standard method is lower than that of the present invention. Furthermore, the industry standard method requires a longer measurement time of 30 minutes, which is longer than the measurement time of the present invention.

[0105] Therefore, after comparison, it can be concluded that compared with the conventional standard detection method, the present invention has higher accuracy and shorter detection time.

[0106] The third comparison

[0107] The test was conducted using non-iodized salt as in Example 4, but the determination method was the o-phenanthroline spectrophotometric method specified in the light industry standard "Determination of the Anticaking Agent Ammonium Ferric Citrate in Edible Salt" (QB / T 5776). The test steps were carried out in accordance with the test steps specified in the standard. The test results are as follows:

[0108] Table 8 Spectrophotometric determination of non-iodized salt samples by o-phenanthroline and recovery of spiked salt (n=6)

[0109]

[0110]

[0111] After analyzing the data in Table 8, it can be known that, compared with the inventive method, the deviation value of the recovery of spiked recovery is larger when the method adopting industry standard regulations is detected without iodized salt sample. That is, the accuracy of its testing result is lower than the accuracy of the inventive method when the method adopting industry standard regulations is adopted. Simultaneously, the determination time is 30 minutes, which is longer than the determination time of the present invention.

[0112] Therefore, after comparison, it can be concluded that compared with the conventional standard detection method, the present invention has higher accuracy and shorter detection time.

[0113] It should be noted that although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic creative concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0114] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for detecting the content of ammonium ferric citrate in salt using nitrogen-doped carbon dot nanozymes, characterized in that: The method The following steps are involved: S1, prepare standard solution Preparation of ammonium ferric citrate standard sample solution: within the concentration range of 0-6 μg / mL, take nine groups of ammonium ferric citrate standard solutions with different concentrations according to an arbitrary concentration gradient and add them to nine colorimetric tubes respectively; then add nitrogen-doped carbon dot nanozyme solution, 2,2'-hydrazino-bis-3-ethylbenzothiazoline-6-sulfonic acid and H2O2 to the colorimetric tubes respectively, dilute to 4 mL with pH 7.0 phosphate buffer solution, mix well, and let stand for 5-10 minutes to obtain nine groups of different standard sample solutions. Blank standard solution: Add nitrogen-doped carbon dot nanozyme solution, 2,2'-hydrazino-bis-3-ethylbenzothiazoline-6-sulfonic acid, and H2O2 to a colorimetric tube, dilute to 4 mL with pH 7.0 phosphate buffer solution, mix well, and let stand for 5-10 minutes to obtain a blank standard sample solution. S2, prepare standard curve Determine the absorbance of the standard solution at the maximum absorption wavelength, use the concentration of the ammonium ferric citrate standard solution as the horizontal axis and the corresponding absorbance difference ΔA as the vertical axis, ΔA=A-A0, draw a standard curve, and calculate the standard curve regression equation. A is the absorbance of the ammonium ferric citrate standard sample solution at the maximum absorption wavelength; A0 is the absorbance of the blank standard solution at the maximum absorption wavelength. S3, detection of the content of ammonium ferric citrate in salt samples Prepare salt sample solution: prepare salt sample solution with a concentration of 0.2 g / mL; Take 0.5 mL of the salt sample solution, then add nitrogen-doped carbon dot nanozyme solution, 2,2'-hydrazino-bis-3-ethylbenzothiazoline-6-sulfonic acid and H2O2 into a colorimetric tube, dilute to 4 mL with pH 7.0 phosphate buffer solution, mix well, let stand for 5 to 10 minutes, measure the absorbance at the maximum absorption wavelength, and calculate the content of ammonium ferric citrate in the sample using the standard curve regression equation.

2. The method according to claim 1, wherein The concentration of the nitrogen-doped carbon dot nanozyme solution is 1 mg / mL, and the added amount is 50-100 μL; the concentration of the 2,2'-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid is 5 mmol / L, and the added amount is 50-100 μL; the concentration of the H2O2 is 50 mmol / L, and the added amount is 50-100 μL.

3. The method according to claim 2, wherein The amount of the nitrogen-doped carbon dot nanozyme solution added was 100 μL; the amount of the 2,2'-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid added was 100 μL; and the amount of the H2O2 added was 100 μL.

4. The method according to claim 1, wherein The maximum absorption wavelength is 416 nm.

5. A method for preparing the nitrogen-doped carbon dot nanozyme solution according to the method of claim 1, characterized in that: The preparation method comprises the following steps: Dissolve 20-30 mg of octopamine and 2.0-2.5 g of citric acid in 30-40 mL of deionized water, then add 50-100 μL of ethylenediamine, and ultrasonically treat for 20-30 min to obtain a mixed reagent; then perform microwave digestion on the mixed reagent, cool to room temperature after the reaction is completed to obtain a suspension; filter the suspension and then centrifuge to obtain a supernatant, and dry the supernatant to obtain nitrogen-doped carbon dot nanozyme; finally, add deionized water to obtain a nitrogen-doped carbon dot nanozyme solution.

6. The preparation method according to claim 5, wherein The specific operation of the microwave digestion is: the mixed reagent is subjected to 850W microwave to raise the reaction temperature to 180°C within 1 minute, and the reaction is continued for 1 to 2 hours.

7. The preparation method according to claim 5, wherein The specific operation of the filtration is to filter the suspension using a filter membrane with a pore size of 22 μm.

8. The preparation method according to claim 5, wherein The centrifugal speed is 8000-10000 r / min, and the time is 5-10 min.

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