A dual-mode MnO2 / Fe, B-CDs peroxidase method for detecting nitrite

Abstract

The present invention discloses a dual-mode method for detecting nitrite by using MnO2 / Fe,B-CDs with peroxidase-like activity. In the present invention, iron and boron co-doped carbon dots (Fe,B-CDs) are synthesized by a microwave method and used as a reducing agent to prepare MnO2 / Fe,B-CDs nanomaterials. MnO2 / Fe,B-CDs have peroxidase-like activity and can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 to produce blue oxTMB. On the one hand, when nitrite is added together with TMB and H2O2, the peroxidase-like activity of the nanozyme can be enhanced, deepening the blue color of the system. On the other hand, after MnO2 / Fe,B-CDs oxidize TMB and H2O2 to produce blue oxTMB, when nitrite is added, a diazo reaction occurs between nitrite and oxTMB, forming yellow diazonium salt. Thus, a new dual-color and dual-mode detection method for nitrite at wavelengths of 654 nm and 440 nm is established. Applying this method to the detection and analysis of nitrite in food and fermented food has the characteristics of accuracy, high sensitivity, strong specificity, simple operation and rapidity.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis and detection, and specifically relates to a method for detecting nitrite by dual-mode MnO2 / Fe, B-CDs peroxidase. Background Art

[0002] Nitrite (NO2 - ) is a food additive widely used in food to preserve freshness (antioxidant) or inhibit microbial growth (preservative). However, NO2 - The amount of addition is strictly limited, and large amounts of NO2 - Will cause serious harm to human health. - It can react with amides or secondary amines in the stomach to generate carcinogenic N-nitrosamines, and combine with hemoglobin to generate methemoglobin, inducing hypoxia in the body. In order to better ensure the quality and safety of food, it is necessary to accurately measure NO2- in food. - For the detection of NO2, various detection methods including chromatography, electrochemistry and spectroscopy are used. Among these methods, colorimetry is more widely used in the detection of NO2 due to its advantages of low cost, simple and rapid operation. - However, there is also the problem of interference. In order to increase the reliability of data, using optical dual signal output to detect samples is a new research direction.

[0003] Nanozymes are nanomaterials with enzymatic activity. Compared with natural enzymes, they have the advantages of low cost, good stability, high tolerance, and large-scale production. 3,3',5,5'-Tetramethylbenzidine (TMB) is the most commonly used chromogenic substrate to date, which can be used for visualization and absorption spectral analysis. The mechanism of TMB colorimetric reaction depends on the core group - benzidine. In the presence of oxidants, colorless TMB first loses an electron to form a blue electron transfer complex with another TMB molecule. When TMB loses another electron, a yellow oxidized state TMB (oxTMB) is formed. TMB has an ideal molar absorption coefficient and a low oxidation potential, so it is easily oxidized. For nitrite, the partially oxidized form of nitrogen in the nitrite anion allows its dual viability based on oxidation or reduction detection. Summary of the invention

[0004] The present invention synthesizes iron and boron doped carbon dots (Fe, B-CDs) by microwave method, and uses the carbon dots as reducing agents to prepare MnO2 / Fe, B-CDs nanomaterials. MnO2 / Fe, B-CDs has peroxidase-like activity and can oxidize the blue oxTMB produced by TMB and H2O2 system. On the one hand, the addition of nitrite together with TMB and H2O2 can enhance the peroxidase-like activity of the nanozyme and deepen the blue color of the system. On the other hand, after MnO2 / Fe, B-CDs oxidizes the blue oxTMB produced by TMB and H2O2 system, nitrite is added to make nitrite react with oxTMB to form yellow diazonium salt. Thus, a new dual-color dual-mode detection method of nitrite at wavelengths of 654 nm and 440 nm is established. The method is applied to the detection and analysis of nitrite in food and fermented food, and has the characteristics of accuracy, high sensitivity, strong specificity, simple operation, rapidity, etc.

[0005] The method for detecting nitrite by the dual-mode MnO2 / Fe, B-CDs peroxidase of the present invention is as follows:

[0006] (1) Add 0.5-1.0 g FeCl3·6H2O, 0.1-0.2 g MgB2, 2.0-2.5 g citric acid and 100-150 μL ethylenediamine to 30-50 mL deionized water, and after ultrasonic treatment for 20-30 min, transfer the mixed solution to a polytetrafluoroethylene autoclave, place it in a microwave digester and heat it to 170-190°C for 1-2 h. After the reaction is completed, cool it naturally to room temperature, remove large particles with a 0.22 μm filter membrane, and then centrifuge it at high speed to remove the supernatant, which is Fe,B-CDs;

[0007] (2) Add 2-3 mL 0.01 mol / L KMnO4 to 15-20 mL deionized water under stirring, heat to boiling in an oil bath at 130-140°C, add 200-300 μL of Fe, B-CDs prepared in step (1) to the boiling aqueous solution, heat and stir for 15-20 min, stop heating and cool the resulting mixture to room temperature under continuous stirring, and dry in vacuum to obtain MnO2 / Fe, B-CDs;

[0008] (3) Add MnO2 / Fe,B-CDs nanozyme, 3,3',5,5'-tetramethylbenzidine (TMB), H2O2 and different gradient concentrations of nitrite solution to pH 4.0 acetic acid-sodium acetate buffer solution, mix well, incubate for 10-15 min, measure the absorbance at a wavelength of 654 nm, establish a quantitative relationship between absorbance and nitrite concentration, draw a standard curve, and obtain a regression equation; In addition, add MnO2 / Fe,B-CDs nanozyme, TMB and H2O2 to pH 4.0 acetic acid-sodium acetate buffer solution, mix well, incubate for 10-15 min, then add different gradient concentrations of nitrite solution, react for 5-10 min, measure the absorbance at a wavelength of 440 nm, establish a quantitative relationship between absorbance and nitrite concentration, draw a standard curve, and obtain a regression equation;

[0009] (4) Determination in the sample: add pH 4.0 acetic acid-sodium acetate buffer solution, MnO2 / Fe, B-CDs nanozyme, TMB, and H2O2 to the sample solution to be tested, mix well, incubate for 10-15 min, measure the absorbance at a wavelength of 654 nm, and substitute the absorbance into the regression equation in step (3) to obtain the nitrite content in the sample; at the same time, add MnO2 / Fe, B-CDs nanozyme, TMB and H2O2 to pH 4.0 acetic acid-sodium acetate buffer solution, mix well, incubate for 10-15 min, and then add the sample solution to be tested, react for 5-10 min, measure the absorbance at a wavelength of 440 nm, and substitute the absorbance into the regression equation in step (3) to obtain the nitrite content in the sample.

[0010] The concentration of the MnO2 / Fe,B-CDs peroxidase solution is 1 mg / mL, and the amount added is 100-150 μL; the concentration of TMB is 5 mmol / L, and the amount added is 100-200 μL; the concentration of H2O2 is 50 mmol / L, and the amount added is 100-200 μL.

[0011] The advantages of the present invention are:

[0012] 1. The present invention utilizes the peroxidase-like activity of the synthesized MnO2 / Fe, B-CDs to oxidize the blue oxTMB produced by the TMB and H2O2 system. On the one hand, the addition of nitrite together with TMB and H2O2 can enhance the peroxidase-like activity of the nanozyme, which is attributed to NO2 -The generated ·NO2 free radicals increase the generation of •OH and deepen the blue color of the system. On the other hand, when MnO2 / Fe,B-CDs oxidizes TMB and H2O2 system to produce blue oxTMB, nitrite is added to make nitrite react with oxTMB to form yellow diazonium salt. Thus, a new dual-color dual-mode detection method for nitrite at wavelengths of 654 nm and 440 nm was established.

[0013] 2. Optical dual-mode detection increases data reliability and reduces the defects of optical detection itself. At the same time, the established dual-mode detection method has high detection sensitivity, reaching 0.01 mg / L and 0.005 mg / L respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a transmission electron microscope TEM image of the MnO2 / Fe,B-CDs nanozyme prepared in Example 1;

[0015] Figure 2 The MnO2 / Fe, B-CDs nanozyme, MnO2 / Fe, B-CDs nanozyme + NO2 in Example 1 - UV-vis absorption spectra of catalytic oxidation peroxidase chromogenic substrate TMB and H2O2;

[0016] Figure 3 After the MnO2 / Fe,B-CDs nanozyme catalyzes the oxidation of the peroxidase chromogenic substrate TMB and H2O2 in Example 1, NO2 is added - UV-vis absorption spectrum;

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

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

[0019] Figure 6 MnO2 / Fe, B-CDs+TMB+H2O2+NO2 in Example 1 - Detection of NO2 - Absorption spectra (left) and linear fitting curves (right) at 0.05~17.5 mg / L;

[0020] Figure 7 oxTMB+NO2 in Example 1 - Detection of NO2 - Absorption spectrum (left) and linear fitting curve (right) at 0.033~6.67 mg / L;

[0021] Figure 8 Detection of NO2 for MnO2 / Fe,B-CDs - The results of selection specificity, the left picture is MnO2 / Fe, B-CDs+TMB+H2O2+NO2 - System detection NO2 - Interference diagram of oxTMB+NO2 - Detection of NO2 - The interference diagram. 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. The methods in the embodiments are conventional methods or detection methods unless otherwise specified, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified.

[0023] Example 1: Determination of nitrite content in vegetable samples

[0024] 1. Preparation of iron and boron doped carbon dots (Fe,B-CDs)

[0025] 0.5 g FeCl3·6H2O, 0.1 g MgB2, 2.0 g citric acid and 100 μL ethylenediamine were added to 30 mL deionized water. After ultrasonic treatment for 20 min, the mixed solution was transferred to a polytetrafluoroethylene high-pressure reactor and placed in a microwave digester and heated to 170°C for 2 h. After the reaction was completed, it was naturally cooled to room temperature, and large particles were removed with a 0.22 μm filter membrane. The mixture was centrifuged at 10,000 rpm for 15 min, and the supernatant was taken out, which was Fe,B-CDs.

[0026] 2. Preparation of MnO2 / Fe,B-CDs nanozymes

[0027] 2-3 mL 0.01 mol / L KMnO4 was added to 15-20 mL deionized water under stirring conditions, and heated to boiling in an oil bath at 130-140°C. 200-300 μL of Fe, B-CDs prepared in step (1) was added to the boiling aqueous solution. After heating and stirring for 15-20 min, the heating was stopped and the resulting mixture was cooled to room temperature under continuous stirring and vacuum dried to obtain MnO2 / Fe, B-CDs. The prepared MnO2 / Fe, B-CDs nanozyme was analyzed by transmission electron microscopy (TEM). The results are shown in FIG. Figure 1 ,The results showed that the synthesized MnO2 / Fe,B-CDs nanozymes were aggregated and amorphous.

[0028] 3. Determination of MnO2 / Fe, B-CDs nanozyme activity

[0029] 100 μL of 1 mg / mL MnO2 / Fe,B-CDs nanozyme solution or 100 μL of 1 μg / mL NaNO2+100 μL of 1 mg / mL nanozyme MnO2 / Fe,B-CDs, 100 μL of 5 mmol / L TMB, 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, incubated at room temperature for 10 min, and the absorbance was measured at 654 nm using a UV-visible spectrophotometer. Each sample was measured 3 times and the average value was taken. The results are shown in Figure 2 ; As can be seen from the figure, MnO2 / Fe, B-CDs nanozyme exhibits good peroxidase activity, and the addition of NaNO2 will increase the enzyme activity of MnO2 / Fe, B-CDs nanozyme.

[0030] 4. NO2 - Diazotization reaction with oxTMB

[0031] At the same time, 100 μL of 1 mg / mL MnO2 / Fe,B-CDs nanozyme solution, 100 μL of 5 mmol / L TMB, 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, incubated at room temperature for 10 min, and then 100 μL of 1 μg / mL NaNO2 was added. The reaction lasted for 5 min, and the absorbance was measured at 440 nm using a UV-visible spectrophotometer. Each sample was measured 3 times and the average value was taken. The results are shown in Figure 2. Figure 3 ; As can be seen from the figure, NO2 - It undergoes diazotization reaction with oxTMB to form a diazonium salt.

[0032] 5. Detection of free radicals

[0033] 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 MnO2 / Fe,B-CDs nanozyme, 100 μL of 5 mg / mL TA, and 100 μL of 50 μmol / 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 400 nm under an excitation wavelength of 300 nm to explore its catalytic activity. At the same time, 100 μL of 5 μg / mL NaNO2 was added under the same conditions. The results showed that NO2 - The addition of enhanced the fluorescence intensity of the system. Figure 4, MnO2 / Fe,B-CDs+ 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 ).

[0034] 6. NO2 - Working curve production

[0035] Add 2.7 mL of pH 4.0 acetic acid-sodium acetate buffer solution, 100 μL of 1 mg / mL MnO2 / Fe,B-CDs nanozyme, 100 μL of 5 mmol / L TMB, 100 μL of 50 mmol / L H2O2, and nitrite solutions of different gradient concentrations into a centrifugal colorimetric tube, mix well, incubate for 10 min, measure the absorbance at a wavelength of 654 nm, and plot the absorbance versus NO2 - Working curve of concentration, see Figure 6 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1.

[0036] Add 2.7 mL of pH 4.0 acetic acid-sodium acetate buffer solution, 100 μL of 1 mg / mL MnO2 / Fe,B-CDs nanozyme, 100 μL of 5 mmol / L TMB, and 100 μL of 50 mmol / L H2O2 to a centrifugal colorimetric tube, mix well, incubate for 10 min, then add different gradient concentrations of nitrite solution, react for 10 min, measure the absorbance at a wavelength of 440 nm, and plot the absorbance versus NO2 - Working curve of concentration, see Figure 7 The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1.

[0037]

[0038] 7. Method specificity investigation

[0039] In order to evaluate the detection system for NO2 - specificity, a series of common cations (Na + , K + , Ca 2+ Mg 2+ , Cu 2+ 、Zn 2+ , Mn 2+ and Cr 3+ ), anions (Cl - 、NO3 - 、CO3 2- 、HCO3 - 、SO4 2- and PO43- ) and possible coexisting substances (urea (UA), glucose (GO), glutamate (Glu)) and phenylalanine (Phe)) were used to detect the specificity of the two detection systems of the present invention (same as above), in which NO2 - The concentration is 5 mg / L, and the concentration of other interfering substances is NO2 - The concentration is 10 times higher, see Figure 8 The results show that only NO2 - There are obvious enhancement effects and diazotization reactions, and other substances have almost no influence. The method of the invention has good selection specificity.

[0040] 8. NO2 in vegetable samples - Determination of

[0041] (1) Sample pretreatment: Vegetable samples were pretreated using the reference method (GB-5009.33-2016), i.e., ground into a homogenate. 5.0 g of the homogenate, 12.5 mL of a 50 g / L saturated borax solution, and 150 mL of hot water at about 70°C were added to a 250 mL conical flask, heated in a water bath for 15 min, and then taken out and cooled to room temperature. Then, the solution in the conical flask was quantitatively transferred to a 250 mL volumetric flask, and 5 mL of 106 g / L potassium ferrocyanide and 5 mL of 220 g / L zinc acetate solution were added to precipitate protein and remove the sample color. The solution in the volumetric flask was diluted to the scale with water and allowed to stand for 30 min to remove the surface fat. The supernatant was filtered with filter paper, and 30 mL of the initial filtrate was discarded. The filtrate was used as the sample to be tested;

[0042] (2) Sample determination: Add 2.5 mL of pH 4.0 acetic acid-sodium acetate buffer solution, 100 μL of 1 mg / mL MnO2 / Fe,B-CDs nanozyme, 100 μL of 5 mmol / L TMB, 100 μL of 50 mmol / L H2O2, and 0.5 mL of sample determination solution into a centrifugal colorimetric tube, mix well, incubate for 10 min, measure the absorbance at a wavelength of 654 nm, substitute it into the regression equation, and measure NO2 - The contents are shown in Table 2.

[0043] Add 2.5 mL of pH 4.0 acetic acid-sodium acetate buffer solution, 100 μL of 1 mg / mL MnO2 / Fe,B-CDs nanozyme, 100 μL of 5 mmol / L TMB, and 100 μL of 50 mmol / L H2O2 to a centrifugal colorimetric tube, mix well, incubate for 10 min, then add 0.5 mL of sample solution, react for 10 min, measure the absorbance at a wavelength of 440 nm, and measure NO2 - The contents are shown in Table 2.

[0044] At the same time, the spike recovery test was carried out, and the results are shown in Table 2. The results show that the NO2 - The spike recovery rate was between 94.8% and 105.0%, and the RSD was less than 3% (n=6). In order to verify the accuracy of the detection results of the invented method, the national standard method (GB-5009.33-2016) was also used to analyze the nitrite in vegetables every day.

[0045]

[0046] Example 2: NO2 in kimchi samples - Determination of

[0047] 1. Preparation of iron and boron doped carbon dots (Fe,B-CDs)

[0048] 1.0 g FeCl3·6H2O, 0.2 g MgB2, 2.5 g citric acid and 150 μL ethylenediamine were added to 50 mL deionized water. After ultrasonic treatment for 30 min, the mixed solution was transferred to a polytetrafluoroethylene high-pressure reactor and placed in a microwave digester and heated to 180°C for 2 h. After the reaction was completed, it was naturally cooled to room temperature, and large particles were removed with a 0.22 μm filter membrane. The mixture was centrifuged at 8000 rpm for 20 min and the supernatant was taken out, which was Fe,B-CDs.

[0049] 2. Preparation of MnO2 / Fe,B-CDs nanozymes

[0050] 3 mL of 0.01 mol / L KMnO4 was added to 20 mL of deionized water under stirring conditions, and the mixture was heated to boiling in an oil bath at 140°C. 300 μL of Fe, B-CDs prepared in step (1) was added to the boiling aqueous solution. After heating and stirring for 20 min, the heating was stopped and the mixture was cooled to room temperature under continuous stirring and dried in vacuum to obtain MnO2 / Fe, B-CDs.

[0051] 3. NO2 - Working curve preparation: same as Example 1.

[0052] 4. NO2 in kimchi and sausage samples - Determination of

[0053] (1) Sample pretreatment: Accurately weigh 1.0 g of sausage and kimchi, respectively, and place them in 10 mL centrifuge tubes. Then, add 8 mL of deionized water, and ultrasonicate the mixed solution for 1 h to separate the nitrite. After standing for 20 min, take 1.8 mL of the supernatant and add 30 mg of activated carbon powder. The activated carbon powder can adsorb colored substances, metal ions and organic compounds. After standing for 30 min, centrifuge at 10,000 rpm for 15 min. Finally, remove the supernatant and filter through a 0.22 μm filter membrane to obtain the sample test solution.

[0054] (2) NO2 - Determination of NO2 in kimchi and sausage samples: Same as in Example 1. - The contents at 654 nm and 440 nm were 1.5 mg / kg and 1.7 mg / kg, 3.5 mg / kg and 3.7 mg / kg respectively.

Claims

1. A dual-mode MnO2 / Fe, B-CDs peroxidase method for detecting nitrite, characterized in that: The following steps are involved: (1) 0.5-1.0 g FeCl3·6H2O, 0.1-0.2 g MgB2, 2.0-2.5 g citric acid and 100-150 μL ethylenediamine were added to 30-50 mL deionized water, and after ultrasonic treatment for 20-30 min, the mixed solution was transferred to a polytetrafluoroethylene high-pressure reactor, placed in a microwave digester and heated to 170-190°C for 1-2 h. After the reaction was completed, it was naturally cooled to room temperature, and large particles of impurities were removed by a 0.22 μm filter membrane. After high-speed centrifugation, the supernatant was taken out, which was Fe, B-CDs; (2) adding 2-3 mL of 0.01 mol / L KMnO4 to 15-20 mL of deionized water under stirring conditions, heating to boiling in an oil bath at 130-140° C., adding 200-300 μL of Fe, B-CDs prepared in step (1) to the boiling aqueous solution, heating and stirring for 15-20 min, stopping heating and cooling the resulting mixture to room temperature under continuous stirring, and vacuum drying to obtain an iron-boron doped carbon dot composite MnO2 nanomaterial MnO2 / Fe, B-CDs; (3) Add MnO2 / Fe,B-CDs nanozyme, 3,3',5,5'-tetramethylbenzidine, H2O2 and different gradient concentrations of nitrite solution to pH 4.0 acetic acid-sodium acetate buffer solution, mix well, incubate for 10-15 minutes, measure absorbance at a wavelength of 654 nm, establish a quantitative relationship between absorbance and nitrite concentration, draw a standard curve, and obtain regression equation 1; in addition, add MnO2 / Fe,B-CDs nanozyme, 3,3',5,5'-tetramethylbenzidine and H2O2 to pH 4.0 acetic acid-sodium acetate buffer solution, mix well, incubate for 10-15 minutes, then add different gradient concentrations of nitrite solution, react for 5-10 minutes, measure absorbance at a wavelength of 440 nm, establish a quantitative relationship between absorbance and nitrite concentration, draw a standard curve, and obtain regression equation 2; (4) Determination in the sample: add pH 4.0 acetic acid-sodium acetate buffer solution, MnO2 / Fe, B-CDs nanozyme, 3,3',5,5'-tetramethylbenzidine, and H2O2 to the sample solution to be tested, mix well, incubate for 10-15 minutes, measure the absorbance at a wavelength of 654 nm, and substitute the absorbance into the regression equation 1 in step (3) to obtain the nitrite content in the sample; at the same time, add MnO2 / Fe, B-CDs nanozyme, 3,3',5,5'-tetramethylbenzidine and H2O2 to the pH 4.0 acetic acid-sodium acetate buffer solution, mix well, incubate for 10-15 minutes, and then add the sample solution to be tested, react for 5-10 minutes, measure the absorbance at a wavelength of 440 nm, and substitute the absorbance into the regression equation 2 in step (3) to obtain the nitrite content in the sample.

2. The method for detecting nitrite by dual-mode MnO2 / Fe, B-CDs peroxidase according to claim 1, characterized in that: The concentration of MnO2 / Fe,B-CDs peroxidase solution is 1 mg / mL, and the amount added is 100-150 μL; the concentration of 3,3',5,5'-tetramethylbenzidine is 5 mmol / L, and the amount added is 100-200 μL; the concentration of H2O2 is 50 mmol / L, and the amount added is 100-200 μL.

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