Method for multi-mode detection of nitrite based on non-metallic nanocatalyst enhanced colorimetric and electrochemical signal amplification and application thereof

Abstract

The application provides a method for multi-mode detection of nitrite based on non-metallic nanocatalyst enhanced colorimetric and electrochemical signal amplification, and belongs to the technical field of detection. The application comprises the following steps: step 1), preparation of P-N-C non-metallic nanoenzyme; step 2), construction of a multi-mode sensor; step 3), obtaining the linear correlation of the absorbance value of NO2 ‑ under different concentrations obtained in step 2) with the absorbance value of NO2 ‑ at 650 and 445 nm in a colorimetric mode; and the linear correlation of the ratio of the electric signal generated by the diazotization reaction of NO2 ‑ and 3, 3', 5, 5'-tetramethylbenzidine (TMB) to the reduced electric signal of the TMB solution itself in an electrochemical mode. The application has high catalytic activity by using P-N-C non-metallic nanoenzyme, and the synthesis cost is greatly reduced because the non-metallic nanoenzyme does not contain metal components and is green and environmentally friendly. The application of the non-metallic nanoenzyme to the multi-mode sensor can provide high-sensitivity catalytic signals for the detection of nitrite.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a multi-mode detection method for nitrite based on non-metallic nanocatalyst-enhanced colorimetry and electrochemical signal amplification, as well as the application of this detection method in detecting nitrite in cured meat, sausages, and dried shrimp. Background Technology

[0002] Nitrite (NO2) - Nitrites are nitrogen dioxide salts found in food and are widely used in the food industry as preservatives and colorants. However, excessive addition of nitrites can lead to health problems, and long-term excessive intake may cause cancer. Therefore, sensitive detection of NO2 in food is crucial. - The content of NO2 is very important for human health. my country also targets the levels of NO2 in different types of food. - Standard testing methods and strict limits have been established for the content.

[0003] Nanozymes, as a novel type of nanomaterial with enzyme-like catalytic activity, have attracted widespread attention. Compared with natural enzymes, they have stronger stability, excellent catalytic performance, and lower cost.

[0004] Currently, a method using Mn3O4 nanoparticles to catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue TMBox has been developed, utilizing a dual-mode diazotization reaction for NO2 detection. - The experiment showed that this method achieves a relatively small detection range (0-300) μM and is highly dependent on the equipment.

[0005] There is currently a method for detecting nitrite in food based on Fe single-atom nanozymes (patent number 2022115943309). This invention relates to the preparation of a dodecahedral Fe single-atom nanozyme and its application in nitrite detection. The detection reagent consists of a catalytic substrate (chromogenic) reagent, a catalyst, and a buffer solution. The method involves: adding the catalyst onto test paper to form a test strip; then preparing a solution of the catalytic substrate (chromogenic agent) and the collected sample to be tested; and then dropping these solutions onto dried test paper and incubating for a certain period of time until the test strip changes color. Finally, an app (ColorDetector) on a smartphone is used to read the RGB value of the test strip color. This patent uses a single-mode detection method, and the Fe single-atom nanozyme is a metal nanozyme, resulting in higher production costs.

[0006] Current technologies focus on metal nanocatalysts, but do not involve the use of non-metal nanocatalysts to enhance colorimetric and electrochemical signal amplification in multi-mode nitrite detection methods.

[0007] Currently with NO2 -Detection methods are constantly evolving, with techniques such as colorimetry, electrochemical methods, fluorescence methods, high-performance liquid chromatography (HPLC), and Raman spectroscopy becoming increasingly mature. Among these, HPLC offers high sensitivity and good reproducibility, but it is costly and complex to operate. Fluorescence methods have low accuracy and are not suitable for rapid on-site detection. These methods rely on a single mode of detection, making them relatively unstable and susceptible to interference from external conditions, which can lead to inaccurate results. Summary of the Invention

[0008] In view of this, in order to solve the problem of NO2 in the existing technology - To address the technical problems of low accuracy and high production cost of detection methods, this invention provides a multi-mode detection method for nitrite based on non-metallic nanocatalysts that enhance colorimetric and electrochemical signal amplification. By employing PNC non-metallic nanozymes, which not only possess high catalytic activity but also contain no metal components, the synthesis cost is greatly reduced, making it environmentally friendly. Applying it to a multi-mode sensor can provide a highly sensitive catalytic amplification signal for nitrite detection.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A multi-mode detection method for nitrite based on non-metallic nanocatalysts to enhance colorimetric and electrochemical signal amplification includes the following steps:

[0011] Step 1) Preparation of PNC non-metallic nanozymes

[0012] Step 2) Construction of multi-mode sensors

[0013] Multiple solutions of 3,3',5,5'-tetramethylbenzidine (TMB) were added to a mixed solution of the PNC nonmetallic nanozyme suspension prepared in step 1) and NaAc-HAc buffer and incubated. Then, different concentrations of NO2 were added. - The solution was added to several of the above mixtures, and the reactions were carried out at room temperature. After the reactions were completed, NO2 concentrations were obtained by colorimetry (UV-vis), electrochemical method (DPV), and smartphone photography, respectively. - Experimental data related to absorbance, peak current, and RGB values ​​under different conditions;

[0014] Step 3) Based on the different concentrations of NO2 obtained in Step 2) - Experimental data related to absorbance values ​​were obtained in colorimetric mode for NO2. - The linear correlation between the ratios of absorbance values ​​at concentrations of 650 and 445 nm; and

[0015] NO2 in electrochemical mode - Concentration and NO2 -The linear correlation between the ratio of the generated electrical signal and the electrical signal reduced by the TMB solution itself.

[0016] Preferably, in step 1), PNC non-metallic nanozymes are prepared using green tea as raw material and NaH2PO2 as phosphorus source.

[0017] Preferably, the preparation method of PNC nonmetallic nanozymes is as follows:

[0018] Washed and dried green tea was soaked in a 7% potassium hydroxide solution for 6 hours, then carbonized at 400°C for 2 hours. 0.9g of activated carbon and 55mg of NaH2PO2·H2O were added, along with 10ml of deionized water. The mixture was stirred rapidly at room temperature for 6 hours. The reaction solution was immediately frozen with liquid nitrogen and freeze-dried for nearly 12 hours. The resulting dark black material was heat-treated at 800°C for 2 hours in the presence of nitrogen in CO(NH2)2. It was then washed with 0.5M hydrochloric acid and deionized water to remove free metals and impurities. The synthesized product was dried in a vacuum oven at 60°C for 13 hours. The final material was defined as PNC nonmetallic nanozyme.

[0019] Preferably, in step 3), NO2 in colorimetric mode - The linear relationship between the concentration and the ratio of absorbance values ​​at 650 and 445 nm is y = 1.9219x + 5.6892, LOD = 0.24 μM, where y is the ratio of absorbance at 650 and 445 nm, and x is the concentration of NO2. - The logarithm of concentration.

[0020] Preferably, in step 3), NO2 is used in electrochemical mode. - Concentration and NO2 - The linear relationship between the ratio of the generated electrical signal and the electrical signal reduced by the TMB solution itself is y = 0.0051 + 0.0041x, LOD = 0.21 μM, where y is NO2. - The ratio of the increasing electrical signal to the decreasing electrical signal in TMB, where x is NO2. - concentration.

[0021] Preferably, in step 2), the colorimetric method uses a smartphone's high-definition camera to capture image information and uses RGB data analysis to obtain colorimetric data.

[0022] Preferably, it also includes a paper chip for NO2 detection. - It includes the following steps:

[0023] (1) Add PNC non-metallic nanozyme solution to the paper chip and dry it;

[0024] (2) Add TMB solution and buffer solution to the test strip and react for 15 min;

[0025] (3) Add NO2 to the paper chip - The test solution is used to determine NO2 based on color change. - content.

[0026] Preferably, step 2) involves constructing the multi-mode sensor as follows:

[0027] Add 25 μL of TMB solution (3 mM) to a mixture of 25 μL of PNC suspension (0.24 mg / mL) and 75 μL of NaAc-HAc buffer (pH 4.0, 0.2 M), incubate at 35 °C for 15 min, and then add 25 μL of NO2 at different concentrations. - The solution is reacted at room temperature.

[0028] This invention also provides the application of the above-mentioned multi-mode detection method for nitrite based on non-metallic nanocatalyst-enhanced colorimetry and electrochemical signal amplification in the detection of nitrite in food.

[0029] Preferably, the food includes at least one of cured meat, dried shrimp, and sausage.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides a multi-mode detection method for nitrite based on non-metallic nanocatalysts to enhance colorimetric and electrochemical signal amplification. By using PNC non-metallic nanozymes, it not only has high catalytic activity but also does not contain metal components, which greatly reduces the synthesis cost and is environmentally friendly. Applying it to a multi-mode sensor can provide a highly sensitive catalytic amplification signal for nitrite detection.

[0032] This invention provides a multi-mode detection method for nitrite based on non-metallic nanocatalysts enhanced colorimetry and electrochemical signal amplification. The multi-mode detection allows for cross-verification, improving the reliability of the analytical results. The colorimetric method enables visual detection and offers rapid reaction speed, meeting the needs of rapid on-site detection. The electrochemical method boasts high sensitivity and a low detection limit. The widespread use of smartphones with built-in high-definition cameras allows for capturing reaction images of different NO2 concentrations. - It can produce different color reactions, therefore, by analyzing its RGB values ​​through image processing, NO2 can be detected. - The goal is to achieve detection of NO2 without relying on large instruments and equipment. (Paper chip-assisted detection of NO2) - It also achieves qualitative detection of NO2 through color changes. - The purpose is to achieve this, and its advantages include low production cost and portability. Combining these advantages, multi-mode sensors provide more accurate and reliable detection results and have a wider range of applications. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of a method for detecting nitrite based on PNC nonmetal nanozyme colorimetry and electrochemical sensor;

[0034] Figure 2 This is an optimized pH graph, where, Figure 2 In diagram A, the UV-Vis spectra are at different pH values. Figure 2 Figure B shows the relative activity (%).

[0035] Figure 3 The diagram shows the optimized concentration of PNC nonmetal nanozymes, where... Figure 3 In the image, A represents the UV-Vis spectra at different PNC concentrations. Figure 3 Figure B shows the corresponding relative activity (%).

[0036] Figure 4 The graph shows the optimal reaction temperature, where... Figure 4 In diagram A, the ultraviolet-visible absorption spectra are at different temperatures. Figure 4 Figure B shows the corresponding relative activity (%).

[0037] Figure 5 The graph shows the optimized TMB concentration, where... Figure 5 In the diagram, A represents the UV-Vis spectra at different TMB concentrations. Figure 5 Figure B shows the corresponding relative activity (%).

[0038] Figure 6 The UV-Vis spectra of nitrite at different concentrations in the PN-C+TMB system are shown.

[0039] Figure 7 This is a calibration curve of absorbance ratio (A650 / A445) versus the logarithm of nitrite concentration.

[0040] Figure 8 DPV curves of different concentrations of nitrite in the PN-C+TMB system;

[0041] Figure 9 The ratio of the peak current of DPV (I) Nitrite / I TMB The linear relationship between nitrite concentration and nitrite concentration.

[0042] Figure 10 Colorimetric images of paper sensors with different concentrations of nitrite (0-800 μM);

[0043] Figure 11 The calibration curves are shown for the RGB values ​​of nitrite at different concentrations versus the logarithm of nitrite concentration. The inset shows the corresponding images. Detailed Implementation

[0044] like Figure 1 As shown, this invention provides a multi-mode detection method for nitrite based on non-metallic nanocatalyst-enhanced colorimetry and electrochemical signal amplification. It is a multi-mode nitrite detection sensor based on non-metallic catalyst-enhanced colorimetry and electrochemical signal amplification, the specific principle of which is as follows:

[0045] This invention utilizes PNC nonmetal nanozymes as an electrochemical signal amplification and catalyst for the reaction. PNC nonmetal nanozymes can specifically catalyze colorless 3,3',5,5'-tetramethylbenzidine (TMB) to form blue TMB oxide (TMBox). NO2 is then added. - Subsequently, it further oxidizes TMBox, undergoing a diazotization reaction. UV-Vis analysis revealed a significant decrease in absorbance at 650 nm and a significant increase in absorbance at 445 nm. In the electrochemical sensor, using the DPV method, the original peak value of TMB (0.43 V) decreased, and a new peak (NO2) appeared. - A peak (0.72V) is generated. Observing the experimental phenomena, this reaction proceeds with the reaction involving NO2. - An increase in NO2 concentration produces three distinct color changes: blue, green, and yellow. Therefore, a smartphone's high-definition camera can capture image information (different NO2 concentrations). - (Color changes at different concentrations) and RGB value analysis of the colors. Detection was performed at different concentrations, and standard curves were established for multiple modes to achieve the detection of samples with unknown concentrations. Similarly, NO2 at different concentrations was detected in the paper chip. - The different color changes produced can also achieve the purpose of qualitative detection.

[0046] like Figure 1 As shown, the multi-mode detection method for nitrite based on non-metallic nanocatalyst-enhanced colorimetry and electrochemical signal amplification provided by the present invention includes the following steps:

[0047] Step 1) Preparation of PNC non-metallic nanozymes

[0048] Step 2) Construction of multi-mode sensors

[0049] Multiple solutions of 3,3',5,5'-tetramethylbenzidine were added to a mixed solution of the PNC nonmetallic nanozyme suspension prepared in step 1) and NaAc-HAc buffer and incubated. Then, different concentrations of NO2 were added. - The solution was added to several of the above mixtures, and the reactions were carried out at room temperature. After the reactions were completed, NO2 concentrations were obtained by colorimetry (UV-vis), electrochemical method (DPV), and smartphone photography, respectively.- Experimental data related to absorbance, peak current, and RGB values ​​under different conditions;

[0050] Step 3) Based on the different concentrations of NO2 obtained in Step 2) - Experimental data related to absorbance values ​​were obtained in colorimetric mode for NO2. - The linear correlation between the ratios of absorbance values ​​at concentrations of 650 and 445 nm; and

[0051] NO2 in electrochemical mode - Concentration and NO2 - The linear correlation between the ratio of the generated electrical signal and the electrical signal reduced by the TMB solution itself.

[0052] In this invention, in step 1), PNC non-metallic nanozymes are prepared using green tea as raw material and NaH2PO2 as phosphorus source. These nanozymes exhibit better catalytic performance, and their application in sensor construction can achieve better detection results, such as a wider detection range: 1-700 μM (colorimetric), 1-800 μM (electrochemical), 1-850 μM (RGB), and lower detection limits: LOD = 0.24 μM (colorimetric), LOD = 0.21 μM (electrochemical), and LOD = 0.49 μM (RGB).

[0053] In this invention, the preparation method of PNC nonmetal nanozymes is specifically as follows:

[0054] Washed and dried green tea was soaked in a 7% potassium hydroxide solution for 6 hours, then carbonized at 400°C for 2 hours. 0.9g of activated carbon and 55mg of NaH2PO2·H2O were added, along with 10ml of deionized water. The mixture was stirred rapidly at room temperature for 6 hours. The reaction solution was immediately frozen with liquid nitrogen and freeze-dried for nearly 12 hours. The resulting dark black material was heat-treated at 800°C for 2 hours in the presence of nitrogen in CO(NH2)2. It was then washed with 0.5M hydrochloric acid and deionized water to remove free metals and impurities. The synthesized product was dried in a vacuum oven at 60°C for 13 hours. The final material was defined as PNC nonmetallic nanozyme.

[0055] In this invention, in step 3), NO2 in colorimetric mode - The linear relationship between the concentration and the ratio of absorbance values ​​at 650 and 445 nm is y = 1.9219x + 5.6892, LOD = 0.24 μM, where y is the ratio of absorbance at 650 and 445 nm, and x is the concentration of NO2. - The logarithm of concentration, such as Figure 6-7 As shown.

[0056] In this invention, in step 3), NO2 is used in electrochemical mode. -Concentration and NO2 - The linear relationship between the ratio of the generated electrical signal and the electrical signal reduced by the TMB liquid itself is y = 0.0051 + 0.0041x, LOD = 0.21 μM, where y is NO2. - The ratio of the increasing electrical signal to the decreasing electrical signal in TMB, where x is NO2. - Concentration, such as Figure 8-9 As shown.

[0057] like Figure 10-11 As shown, in this invention, in step 2), the colorimetric method uses a smartphone's high-definition camera to capture image information and uses RGB data analysis to obtain colorimetric data;

[0058] It also includes paper chip-assisted detection of NO2. - It includes the following steps:

[0059] (1) Add PNC non-metallic nanozyme solution to the paper chip and dry it;

[0060] (2) Add TMB solution and buffer solution to the test strip and react for 15 min;

[0061] (3) Add NO2 to the paper chip - The test solution is used to determine NO2 based on color change. - content.

[0062] In this invention, a paper chip is used to assist in the detection of NO2. - It enables portable and rapid detection without relying on large instruments and equipment. It has strong anti-interference capabilities and can be applied to the detection of complex real-world samples (such as cured meat, sausages, dried shrimp, etc.).

[0063] In this invention, step 2), the construction of the multi-mode sensor, specifically involves:

[0064] Add 25 μL of TMB solution (3 mM) to a mixture of 25 μL of PNC suspension (0.24 mg / mL) and 75 μL of NaAc-HAc buffer (pH 4.0, 0.2 M), incubate at 35 °C for 15 min, and then add 25 μL of NO2 at different concentrations. - The solution is reacted at room temperature.

[0065] This invention also provides the application of the above-mentioned multi-mode detection method for nitrite based on non-metallic nanocatalyst-enhanced colorimetry and electrochemical signal amplification in the detection of nitrite in food.

[0066] In this invention, the food includes at least one of cured meat, dried shrimp, and sausage.

[0067] The present invention conducts the following performance tests on the detection performance of the multi-mode nitrite detection method based on non-metallic nanocatalyst-enhanced colorimetry and electrochemical signal amplification for detecting nitrite in food, wherein the selected food items are cured meat, sausage, and dried shrimp.

[0068] 1) Preparation of PNC nonmetallic nanozymes

[0069] Washed and dried green tea (GT) was soaked in a 7% potassium hydroxide solution for 6 hours, then carbonized at 400°C for 2 hours. 0.9 g of activated carbon and 55 mg of NaH₂PO₂·H₂O were added, followed by 10 ml of deionized water. The mixture was stirred rapidly at room temperature for 6 hours. The reaction solution was immediately frozen with liquid nitrogen and freeze-dried for approximately 12 hours. The resulting dark-colored material was heat-treated at 800°C for 2 hours in the presence of nitrogen (N₂) in the presence of (CO(NH₂)₂), followed by washing with 0.5 M hydrochloric acid and DI (deionized) water to remove free metals and impurities. The synthesized product was dried in a vacuum oven at 60°C for 13 hours; the final material was defined as PNC nonmetallic nanozyme (PNC).

[0070] 2) Optimization of experimental conditions

[0071] Furthermore, to ensure that the PNC non-metallic nanozyme achieves the best catalytic effect, screening experiments were conducted, as follows:

[0072] Before the experiment, the PNC nonmetal nanozyme was sonicated for 30 min to ensure its uniform dispersion in the solution. The experimental conditions included the pH value of the NaAc-HAc buffer (1.0-8.0), PNC (0.04-0.28 mg / ml), TMB concentration (0.0-4.0 mM), and reaction temperature (25-55℃).

[0073] Through single-factor experiments, while keeping other experimental conditions constant, one experimental condition is changed to optimize the experiment: such as... Figure 2 As shown, keeping other experimental conditions unchanged (PNC concentration 0.2 mg / ml, TMB concentration 2 mM, temperature 30℃), the pH was changed (1-8). It was found that the maximum absorbance value was reached at pH 4, indicating that the PNC nanozyme activity was the greatest at pH 4.

[0074] like Figure 3 As shown, the pH of the reaction solution was controlled at 4, the TMB concentration at 2 mM, and the reaction temperature at 30℃. The concentration of PNC non-metallic nanozyme was varied (0.04-0.28 mg / ml). It was found that the absorbance value reached its maximum at 0.24 mg / ml, so this concentration is the optimal reaction concentration.

[0075] like Figure 4 As shown, the pH of the reaction solution was controlled at 4, the concentration of PNC non-metallic nanozyme was 0.24 mg / ml, the concentration of TMB was 2 mM, and the reaction temperature was changed (25-55℃). It was found that the absorbance value reached the maximum at 35℃, so the enzyme activity was optimal under this condition.

[0076] like Figure 5 As shown, the pH of the reaction solution was controlled at 4, the concentration of PNC non-metallic nanozyme was 0.24 mg / ml, the reaction temperature was 35℃, and the TMB concentration was changed (1-4 mM). It was found that the absorbance value basically did not increase when the TMB concentration was 3 mM, so this concentration was the optimal concentration.

[0077] In summary, the optimal conditions for PNC nonmetallic nanozymes to achieve the best catalytic effect are: pH 4, concentration 0.24 mg / ml, reaction temperature 35℃, and TMB concentration 3 mM.

[0078] 3) Construction of multi-mode sensors

[0079] Under the optimal conditions described above, 25 μL of TMB solution (3 mM) was added to a mixture of 25 μL of PNC suspension (0.24 mg / mL) and 75 μL of NaAc-HAc buffer (pH 4.0, 0.2 M), and incubated at 35 °C for 15 min. Then, 25 μL of NO2 at different concentrations was added. - The solution was reacted at room temperature. UV-Vis was used to scan the absorbance range of 550-750 nm with 5 nm intervals, recording the wavelength data and separately recording the absorbance values ​​at 445 nm and 650 nm. Under the same conditions, in an electrochemical workstation, DPV mode was used to record changes in the electrical signal within the range of 0.1-1.0 V, and separately recording the electrical signal values ​​at 0.43 V and 0.72 V. Simultaneously, the experimental phenomena were photographed with a mobile phone under stable light conditions, and the RGB data were analyzed using image processing tools.

[0080] 4) Determination of standard curve and detection limit

[0081] Under optimal conditions, a multi-mode sensor was used to detect NO2 at different concentrations. - Colorimetric data and electrochemical signals were obtained. In colorimetric mode, NO2... - The concentration showed a linear correlation with the ratio of absorbance values ​​at 650 and 445 nm, with the linear relationship being y = 1.9219x + 5.6892 (LOD = 0.24 μM), where y is the ratio of absorbance at 650 and 445 nm, and x is the concentration of NO2. - Logarithm of concentration; NO2 in electrochemical mode -Concentration and NO2 - The ratio of the generated electrical signal to the electrical signal reduced by TMB itself shows a linear correlation, with the linear relationship being y = 0.0051 + 0.0041x (LOD = 0.21 μM), where y is NO2. - The ratio of the increasing electrical signal to the decreasing electrical signal in TMB, where x is NO2. - concentration.

[0082] When the concentration of NO2 is unknown - When performing the test, under optimal conditions, add 25 μL of TMB solution (3 mM) to a mixture of 25 μL of PNC suspension (0.24 mg / mL) and 75 μL of NaAc-HAc buffer (pH 4.0, 0.2 M), incubate at 35°C for 15 min, and then add an unknown concentration of NO2. - The absorbance value of the solution (A650 / A445) was measured, and then substituted into the linear equation of the colorimetric mode to obtain NO2. - Concentration. Or, under the same conditions, perform electrochemical (DPV) detection to obtain the same concentration as NO2. - The ratio of the NO2 to the TMB electrical signal can also be used to derive the linear equation in the electrochemical mode. - concentration.

[0083] Experimental Example

[0084] Actual sample extraction: Actual samples included sausage, cured meat, and dried shrimp. Three samples, each 10g, were minced using a meat grinder. 12.5mL of saturated borax solution (50mg / mL) and 150mL of hot water at 70℃ were added to each sample, and the mixture was heated in a water bath for 15min, then cooled to room temperature. 5mL of K₄[Fe(CN)₆] solution (106mg / mL) was added to the mixture, and the mixture was shaken well. Then, it was mixed with 5mL of Zn(Ac)₂ solution (220mg / mL) and stirred. After standing for 30min, the mixture was filtered to obtain the supernatant.

[0085] The aforementioned mixture refers to a mixture of 25 μL TMB solution (3 mM) added to 25 μL PNC suspension (0.24 mg / mL) and 75 μL NaAc-HAc buffer (pH 4.0, 0.2 M) under optimal conditions, incubated at 35°C for 15 min, followed by the addition of an unknown concentration of NO2. - The absorbance value of the solution (A650 / A445) was measured, and then substituted into the linear equation of the colorimetric mode to obtain NO2. - Concentration. Or, under the same conditions, perform electrochemical (DPV) detection to obtain the same concentration as NO2. - The ratio of the NO2 to the TMB electrical signal can also be used to derive the linear equation in the electrochemical mode.- concentration.

[0086] The nitrite content in cured meat, sausage, and dried shrimp was determined using the national standard (GB 5009.33-2016), UV-Vis analysis, electrochemical analysis, and RGB mode. The results are shown in Table 1 below:

[0087] Table 1 shows the nitrite content in cured meat, sausage, and dried shrimp determined using the national standard (GB 5009.33-2016), UV-Vis analysis, electrochemical analysis, and RGB mode.

[0088]

[0089] In Table 1, the nitrite content is expressed by national standard (mg / Kg), colorimetric (mg / Kg), and electrochemical (mg / Kg).

[0090] Three samples were tested using a multi-mode detection method, and the data obtained were compared with the national standard (GB5009.33-2016). The RSDs were all less than 5%, indicating that the method has high reliability and sensitivity.

Claims

1. A method for multi-mode detection of nitrite based on non-metallic nanocatalyst-enhanced colorimetric and electrochemical signal amplification, characterized in that, Comprising the following steps: Step 1), P-N-C nonmetal nanoscale enzyme is prepared by taking green tea as raw material and NaH2PO2 as phosphorus source The preparation method of the P-N-C nonmetal nanoscale enzyme is specifically as follows: The cleaned and dried green tea is soaked in a 7% potassium hydroxide solution for 6 hours, then carbonized at 400 DEG C for 2 hours, 0.9 g of activated carbon and 55 mg of NaH2PO2.H2O are added into 10 ml of deionized water, and the mixture is stirred rapidly at room temperature for 6 hours, the reaction solution is immediately frozen with liquid nitrogen, and freeze-dried for nearly 12 hours, the obtained dark black material is heat-treated at 800 DEG C for 2 hours in the presence of CO(NH2)2 under nitrogen atmosphere, then washed with 0.5 M hydrochloric acid and deionized water to clean free metals and impurities, the synthesized product is dried in a vacuum oven at 60 DEG C for 13 hours, and finally the obtained material is defined as P-N-C nonmetal nanoscale enzyme; Step 2), construction of a multi-mode sensor The 3, 3', 5, 5'-tetramethylbenzidine TMB solution was added to the mixed solution of the P-N-C non-metal nanoscale enzyme suspension prepared in step 1) and the NaAc-HAc buffer solution, and then different concentrations of NO2 - solutions were added to the above mixed solutions, and the reaction was carried out at room temperature. After the reaction was completed, the related experimental data of the absorbance value, the current peak value and the RGB value of different concentrations of NO2 - under different concentrations of NO2 were obtained by UV-vis colorimetric method, DPV electrochemical method and smart phone photography, respectively. Step 3) obtaining absorbance values at 650 and 445 nm from different concentrations of NO2 - obtaining experimental data related to the linear correlation of the ratio of the absorbance values at 650 and 445 nm for NO2 - concentrations in colorimetric mode; And In the electrochemical mode NO2 - The linear correlation of the ratio of the electrical signal generated by the diazotization reaction with TMB and the electrical signal of the TMB solution itself is reduced.

2. The method for multi-mode detection of nitrite based on non-metallic nanocatalyst-enhanced colorimetric and electrochemical signal amplification according to claim 1, characterized in that, NO2in the colorimetric mode in step 3) - The linear relationship of the concentration and the ratio of absorbance values at 650 and 445 nm is y = 1.9219x + 5.6892, LOD = 0.24 μΜ, where y is the ratio of absorbance at 650 and 445 nm, and x is the NO2 - concentration.

3. The method for multi-mode detection of nitrite based on non-metallic nanocatalyst-enhanced colorimetric and electrochemical signal amplification according to claim 1, characterized in that, In step 3), NO2 is produced in electrochemical mode. - The linear relationship between the electrical signal generated by the diazotization reaction with TMB and the electrical signal reduced by TMB itself is y = 0.0051 + 0.0041x, LOD = 0.21 μM, where y is NO2. - The ratio of the electrical signal generated by the diazotization reaction with TMB to the electrical signal reduced by TMB, where x represents NO2. - concentration.

4. The method for multi-mode detection of nitrite based on non-metallic nanocatalyst-enhanced colorimetric and electrochemical signal amplification according to claim 1, characterized in that, In step 2), the image information is captured by a high-definition camera of a smart phone, and data analysis and processing are performed by using RGB.

5. The method for multi-mode detection of nitrite based on non-metallic nanocatalyst-enhanced colorimetric and electrochemical signal amplification according to claim 1, characterized in that, Also included are paper chip assisted detection of NO2 - comprising the steps of: (1) P-N-C nonmetal nanoscale enzyme solution is dropped on the paper chip and dried; (2) TMB solution and buffer solution are dropped on the test strip, and reacted for 15 min; (3) Add the solution to be tested containing NO2 on the paper chip, and determine the content of NO2 according to the color change. - - ​​ 6. The method for multi-mode detection of nitrite based on non-metallic nanocatalyst-enhanced colorimetric and electrochemical signal amplification according to any one of claims 1-5, characterized in that, The construction of the multi-mode sensor in step 2) is specifically as follows: To the mixture consisting of 25 μL of P-N-C suspension at a concentration of 0.24 mg / mL and 75 μL of NaAc-HAc buffer at a pH of 4.0 and a concentration of 0.2 M, 25 μL of TMB solution at a concentration of 3 mM was added, and the mixture was incubated at 35°C for 15 min, and then 25 μL of NO2 - solution at different concentrations was added, and the reaction was carried out at room temperature.

7. The application of the method for detecting nitrite by using nonmetal nanocatalyst to enhance colorimetric and electrochemical signal amplification according to any one of claims 1-6 in detecting nitrite in food.

8. Use according to claim 7, characterized in that, The food includes at least one of cured meat, shrimp skin and sausage.

Images