Oxygen-rich vacancy trimanganese tetraoxide nanozyme as well as preparation method and application thereof

Abstract

The present application relates to the technical field of detection, and relates to a preparation method of an oxygen-rich vacancy Mn3O4 nanoscale enzyme, and the method comprises the following steps: step one: adding KMnO4 into deionized water according to a ratio of 0.5-1.5 g:500 ml, and completely dissolving to obtain a mixed solution; step two: adding oleic acid according to a volume ratio of 1:50, and continuously stirring at 25-35 DEG C for 4-6 hours; step three: collecting the precipitate, washing with deionized water and ethanol alternately for 4-6 times, drying at 70-90 DEG C for 8-12 hours, and calcining at 180-220 DEG C for 4-6 hours, so as to obtain an oxygen-rich vacancy Mn3O4 nanoscale enzyme with oxidase activity; and the oxygen-rich vacancy Mn3O4 nanoscale enzyme can detect nitrite rapidly, sensitively and specifically.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to an oxygen-rich vacancy manganese tetroxide nanozyme, its preparation method, and its application. Background Technology

[0002] Nitrite is a salt of nitrite containing nitrite ions (NO2). - Nitrites are ionic compounds, including magnesium nitrite, sodium nitrite, potassium nitrite, calcium nitrite, and silver nitrite. Nitrites are toxic; ingesting even small amounts can cause poisoning, and severe poisoning often leads to death from respiratory failure. Furthermore, nitrites can severely pollute water bodies.

[0003] In existing technologies, nitrite detection methods mainly include the traditional Griess reagent method, chromatography, electrochemical methods, and nanozyme-based detection methods. The Griess reagent method, which develops color through the diazotization reaction of nitrite with aromatic amines, is a classic colorimetric method. Chromatography and electrochemical methods rely on precision instruments and can achieve high-precision quantification. Nanozyme-based detection methods utilize enzymes from nanomaterials to simulate active catalytic chromogenic substrates (such as TMB), combining the specific reaction between the target analyte and the chromogenic product to construct a sensing platform, which is widely used in rapid detection.

[0004] Existing methods for nitrite detection generally have shortcomings. For example, the Griess reagent method has a long reaction time (15-30 minutes), low sensitivity (detection limits are mostly above 5 μM), and is easily affected by pH. Chromatographic and electrochemical methods are complex to operate and expensive to use, making it difficult to meet the needs of on-site real-time detection. Existing nanozyme-based detection methods mostly use substrates such as TMB, which have limited catalytic efficiency and signal amplification capabilities, high detection limits (usually >2 μM), and insufficient resistance to interference from common ions and biomolecules, thus limiting the accuracy of detection in complex food matrices. Summary of the Invention

[0005] The present invention aims to provide a method for preparing oxygen-vacancy manganese tetroxide nanozymes, so as to provide an oxygen-vacancy manganese tetroxide nanozyme that can achieve rapid, sensitive and specific detection of nitrite.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing oxygen-vacancy-enriched manganese tetroxide nanozymes, comprising the following steps:

[0007] Step 1: Add KMnO4 to deionized water at a ratio of 0.5-1.5g:500ml until KMnO4 is completely dissolved to obtain a mixture.

[0008] Step 2: Add oleic acid to the mixture and stir continuously at 25-35°C for 4-6 hours. The volume ratio of oleic acid to deionized water in the mixture is 1:50.

[0009] Step 3: Collect the precipitate, wash it alternately with deionized water and ethanol 4 to 6 times, dry it at 70 to 90°C for 8 to 12 hours, and then calcine it at 180 to 220°C for 4 to 6 hours to obtain oxygen-rich vacancy Mn3O4 nanozyme with oxidase-like activity.

[0010] The principle of this scheme is as follows: KMnO4 dissolves in deionized water, dissociating into MnO4⁻ ions, providing highly active manganese for subsequent reactions. A ratio of 0.5–1.5 g KMnO4 to 500 ml deionized water ensures a suitable concentration of manganese ions in the solution, avoiding runaway reactions due to excessive concentration, and providing sufficient manganese for the formation of oxygen-rich vacancy Mn3O4 nanozymes, laying the material foundation for subsequent crystal growth. Oleic acid is key to regulating the structure of oxygen-rich vacancy Mn3O4 nanozymes. The carboxyl group in the oleic acid molecule can coordinate with manganese ions in the solution, precisely controlling the nucleation and growth rate of crystals through steric hindrance, and inhibiting random aggregation. Stirring at a mild temperature of 25–35°C for 4–6 hours ensures sufficient reaction between oleic acid and the manganese source to form a stable precursor complex, and also introduces potential defect sites into the crystal structure through the surface modification effect of oleic acid, creating conditions for subsequent vacancy formation. The precipitate is washed alternately with deionized water and ethanol, which can effectively remove unreacted K. ⁺ Excessive oleic acid and other impurities are removed to avoid interference with the activity of oxygen-rich vacancy-rich Mn3O4 nanozymes. After drying at 70–90℃ to remove moisture, calcination at 180–220℃ promotes the decomposition and crystal transformation of the precursor, ultimately forming the Mn3O4 crystal structure. This calcination temperature range promotes the decomposition and release of organic components such as oleic acid, leaving oxygen or manganese vacancies in the crystal lattice. The presence of vacancies significantly improves the electron transfer efficiency and the number of surface active sites, thereby endowing Mn3O4 nanomaterials with excellent oxidase-like activity.

[0011] Preferably, as an improvement, in step one, the ratio of KMnO4 to deionized water is 1g:500ml, and KMnO4 is completely dissolved under stirring.

[0012] Research has found that the oxygen-vacancy-enriched Mn3O4 nanozyme prepared in this application can effectively detect nitrite. Therefore, this application also requests protection for the oxygen-vacancy-enriched Mn3O4 nanozyme prepared by the above method.

[0013] This application also seeks protection for the use of oxygen-vacancy-rich Mn3O4 nanozymes in the detection of nitrite.

[0014] Preferably, as an improvement, the nitrite is derived from food or water samples.

[0015] Preferably, as an improvement, the food is ham, sausage, salted egg, bacon, cured meat, smoked meat, canned meat, braised meat products, aquatic products, pickled vegetables, or hot pot soup base.

[0016] Preferably, as an improvement, the water sample is domestic sewage, farmland drainage, irrigation return water, livestock and poultry breeding wastewater, eutrophic water body, groundwater, drinking water or industrial wastewater.

[0017] A method for detecting nitrite using an oxygen-enriched vacancy Mn3O4 nanozyme includes the following steps: In NaAc buffer, a nitrite-containing sample is mixed with the oxygen-enriched vacancy Mn3O4 nanozyme and TOPS for reaction. Visual detection is achieved through color changes in the reaction system, or the RGB values ​​of the reaction system are collected using a smartphone, combined with the absorbance change at 420 nm (ΔA). 420 Quantitative detection was performed using nm.

[0018] Preferably, as an improvement, the NaAc buffer has a pH of 3.0 and a concentration of 10 mM; the concentration of the oxygen-rich vacancy Mn3O4 nanozyme is 70 μg / mL; the concentration of TOPS is 200 μM; and the reaction time is 1 to 30 minutes.

[0019] In the reaction system, oxygen-vacancy-rich Mn3O4 nanozymes catalyze the oxidation of TOPS to generate oxidation products. These oxidation products undergo a diazotization-like reaction with nitrite. The color of the reaction system changes with the concentration of nitrite, and the absorbance at 420 nm changes (ΔA). 420 The concentration of nitrite (nm) showed a linear relationship with the concentration of nitrite. Specifically:

[0020] 1) Take 435 μL of 10 mM NaAc buffer (pH 3.0), add 10 μL of 200 μM TOPS, 20 μL of nitrite samples of different concentrations (with deionized water as a control), and 35 μL of 70 μg / mL oxygen-enriched vacancy Mn3O4 nanozyme, and mix well;

[0021] 2) After reacting at room temperature for 5 minutes, qualitative detection is achieved by visually observing color changes; or quantitative analysis is achieved by using a smartphone to collect the RGB values ​​of the reaction system and combining them with absorbance calculations. When calculating absorbance, .

[0022] A sensor for detecting nitrite includes an oxygen-vacancy-rich Mn3O4 nanozyme, TOPS substrate, NaAc buffer (pH 3.0, 10mM), and a detection device. The detection device includes a visual observation module and a smartphone image analysis module. The smartphone image analysis module outputs quantitative results based on a preset RGB value-nitrite concentration calibration curve. This sensor detects common nitrite cations (Na+, Na ... + K+ Ca 2+ Mg 2+ Cu 2+ etc.), anions (Cl) - SO4 2- PO4 3- It has specific recognition capabilities for molecules such as urea, glucose, and glutamic acid, and has strong anti-interference ability.

[0023] Beneficial effects:

[0024] High sensitivity: The linear detection range is 20-160μM, the detection limit for visual detection is 1.9μM, and the detection limit for smartphone-assisted detection is as low as 0.01μM, which is better than most existing methods;

[0025] High specificity: effective against common cations (Na+) + K + Ca 2+ etc.), anions (Cl) - SO4 2- There was no significant response from (etc.) and biomolecules (urea, glucose, etc.);

[0026] Easy to operate: No complicated instruments are required, the reaction time is short (5 minutes), and rapid on-site detection can be achieved;

[0027] Highly practical: The recovery rate in actual samples such as tap water, salted eggs, and sausages is 79.32%-113.25%, and the deviation from the national standard method (GB 5009.33-2016) is less than 8.5%, making it suitable for food safety production monitoring.

[0028] In this application, the oxygen-vacancy-enriched Mn3O4 nanozyme and the oxygen-vacancy-enriched manganese tetroxide nanozyme are the same substance. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the preparation process of oxygen-rich vacancy Mn3O4 nanozymes.

[0030] Figure 2 Characterization of oxygen-vacancy-rich Mn3O4 nanozymes: (a) SEM, (b) EDS, (c) XRD, (d) XPS, (e) XPS image of Mn 2p, and (f) XPS image of O 1s.

[0031] Figure 3 The figures show the peroxidase (a) and oxidase activities (b) of the oxygen-vacancy-rich Mn3O4 nanozyme, where Mn3O4 refers to the oxygen-vacancy-rich Mn3O4 nanozyme.

[0032] Figure 4The results show the verification of the diazotization-like phenomenon and mechanism; (a) is the investigation of the diazotization-like reaction; (bd) are the verification of the mechanism through free radical quenching experiments, in which p-benzoquinone, tert-butanol, and L-histidine were used to verify the ·O2. ⁻ ·OH and 1 O2, and Mn3O4 in the figure refers to oxygen-rich vacancy Mn3O4 nanozyme.

[0033] Figure 5 The results of cyclic voltammetry verification are shown, where ad represents the response current of Mn3O4, TOPS, Mn3O4+TOPS, and Mn3O4+TOPS+NaNO2, respectively. Mn3O4 in the figure refers to oxygen-rich vacancy Mn3O4 nanozyme.

[0034] Figure 6 The results are for sensor feasibility verification; where (a) 1: Mn3O4, 2: NaNO2+ Mn3O4, 3: TOPS +NaNO2, 4: NaNO2, 5: TOPS; (b) 1: TOPS + Mn3O4, 2: TOPS + Mn3O4+ 50 μM NaNO2, 3: TOPS + Mn3O4+ 100 μM NaNO2, 4: TOPS + Mn3O4+ 150 μM NaNO2, 5: TOPS + Mn3O4+ 200 μM NaNO2.

[0035] Figure 7 The diagram shows the optimization of conditions; where (ae) represents the optimization of pH, buffer concentration, oxygen-rich vacancy Mn3O4 nanozyme concentration, substrate type, and TOPS concentration, respectively. Mn3O4 in the diagram refers to oxygen-rich vacancy Mn3O4 nanozyme.

[0036] Figure 8 The process and analysis results of nitrite detection by the sensor are shown in the figure. (a) is a schematic diagram of nitrite detection, (b) is the sensitivity analysis in visual mode, (c) is the sensitivity analysis in smartphone mode, and (d, e) are the specificity analysis. Mn3O4 in the figure refers to oxygen-vacancy-rich Mn3O4 nanozyme. Detailed Implementation

[0037] The following detailed description illustrates the specific implementation method:

[0038] 1. Preparation and characterization of oxygen-vacancy-enriched Mn3O4 nanozymes

[0039] Preparation process as follows Figure 1As shown: 1.0 g KMnO4 was dissolved in 500 mL of deionized water and stirred at 650 rpm for 30 minutes until completely dissolved; 10 mL of oleic acid was added and stirring was continued at 28 °C for 5 hours; the precipitate was collected, washed 5 times alternately with deionized water and ethanol, dried at 80 °C for 10 hours; and finally calcined at 200 °C for 5 hours to obtain oxygen-rich vacancy Mn3O4 nanozyme.

[0040] Characterization results as follows Figure 2 As shown:

[0041] SEM showed that the oxygen-vacancy-rich Mn3O4 nanozyme was a uniformly dispersed particle with a particle size of about 150 nm and petal-like wrinkles on the surface (Fig. 2a).

[0042] EDS showed that the oxygen-rich vacancy Mn3O4 nanozyme is composed of Mn and O elements (Figure 2b).

[0043] The XRD showed characteristic peaks of Mn3O4 at 2θ = 36.1° and 59.9° (corresponding to crystal planes (211) and (224)), consistent with the standard card JCPDS 97-003-1094 (Fig. 2c).

[0044] XPS confirms the presence of Mn 3+ Mn 4+ and oxygen vacancies (O v (Figure 2d-f).

[0045] 2. Enzyme activity verification

[0046] The verification results are attached. Figure 3 As shown.

[0047] Peroxidase activity: In 435 μL of 20 mM NaAc buffer (pH 3.0), 10 μL of 10 mM TOPS, 20 μL of 30 mM H2O2, and 35 μL of 1 mg / mL oxygen-enriched vacancy Mn3O4 nanozyme were added. After reacting at room temperature for 5 minutes, no characteristic peak appeared at 520 nm (ΔA < 0.05), indicating no peroxidase activity. Figure 3 a).

[0048] Oxidase activity: In 435 μL of 20 mM NaAc buffer (pH 3.0), 10 μL of 10 mM TOPS and 35 μL of 1 mg / mL oxygen-enriched vacancy Mn3O4 nanozyme were added and reacted at room temperature for 5 minutes. A characteristic peak (ΔA = 1.2) was detected at 520 nm using a microplate reader, confirming its oxidase activity. Figure 3 b).

[0049] 3. Verification of the diazotization-like phenomenon and mechanism (as attached) Figure 4 (As shown)

[0050] Reaction phenomena: Mn3O4 catalyzes the oxidation of TOPS to produce a red product (peak at 520 nm). Upon the addition of nitrite, the product turns yellow, and a new peak appears at 420 nm. The color gradually deepens with increasing nitrite concentration. Figure 4 a).

[0051] Mechanism verification:

[0052] Join O2 - After using the quencher P-Benzoquinone (100 mM), the signal at 520 nm and 420 nm decreased by 72% and 68%, respectively. Figure 4 b)

[0053] Adding the ·OH quencher tert-Butanol (100mM) did not significantly change the signal (fluctuation <5%). Figure 4 c)

[0054] join in 1 After using the O2 quencher L-Histidine (100mM), the signal decreased by 65% ​​and 60%, respectively. Figure 4 d);

[0055] Cyclic voltammetry proves that it involves electron transfer ( Figure 5 );

[0056] It was confirmed that the catalytic reaction involved O2. ⁻ , 1 O2 and electron transfer.

[0057] 4. Sensor Construction and Optimization

[0058] Sensor construction

[0059] A dual-mode sensor was constructed, comprising an oxygen-rich vacancy Mn3O4 nanozyme, TOPS substrate, NaAc buffer, and a detection device. The detection device integrates a visual observation module and a smartphone image acquisition and analysis module, which can directly output nitrite concentration results.

[0060] Feasibility verification:

[0061] The control group showed no relevant absorption peaks at 420 nm and 520 nm. Figure 6 a)

[0062] In the experimental group, as the nitrite concentration gradually increased, the solution color changed from purplish-red to yellow. Figure 6 b).

[0063] Optimal conditions:

[0064] Buffer pH=3.0 Figure 7a) Sodium acetate concentration = 10mM ( Figure 7 b); Concentration of oxygen-rich vacancy Mn3O4 nanozyme = 70 μg / mL ( Figure 7 c); Optimal catalytic substrate TOPS ( Figure 7 d); TOPS concentration = 200 μM ( Figure 7 e).

[0065] 6. Sensor performance testing

[0066] Detection principle: The oxygen-rich vacancy Mn3O4 nanozyme has oxidase activity and can catalyze TOPS to turn purple. When nitrite is present, its oxidation product TOPS-ox can react with nitrite to turn yellow, and the color change of the solution is closely related to the concentration of nitrite.

[0067] Sensitivity visual detection: linear in the range of 20-160μM (R 2 =0.98), LOD=1.9μM ( Figure 8 b)

[0068] Smartphone detection (RGB signal): Linear range is the same as above (R 2 =0.99), LOD=0.01μM ( Figure 8 c).

[0069] Specificity: Common ions at 2000 μM (Na+) + K + Ca 2+ Interference signals from molecules such as glucose, urea, etc. and biomolecules (glucose, urea, etc.) are <5%. Figure 8 de).

[0070] Actual sample testing:

[0071] Using tap water, salted eggs, and sausages as actual samples, the samples were processed according to the national standard method (GB 5009.33-2016). Nitrite was then added to the actual samples using a spiked recovery method. The nitrite-containing samples were then mixed with oxygen-rich vacancy Mn3O4 nanozymes and TOPS in NaAc buffer. Visual detection was achieved by observing the color change of the reaction system, or by using a smartphone to collect the RGB values ​​of the reaction system and combining them with the absorbance change at 420 nm (ΔA). 420 The spiking recovery rate was 79.3%-113.2%, and the RSD was <8.5% (Table 1).

[0072] Compared with the national standard method (GB 5009.33-2016) (spectrophotometry), the deviation is <8.5% (Table 2).

[0073] Table 1. Detection of nitrite content in actual samples.

[0074]

[0075] Table 2. Detection of nitrite in actual samples using the national standard method.

[0076]

[0077] 7. Comparative Experiment

[0078]

[0079] Note: The LOD of this invention is 1 / 500 of that of the traditional method, and the detection time is shortened to 1 / 6.

[0080] This invention enhances the activity of oxygen-rich vacancy Mn3O4 nanozymes through vacancy engineering, and combined with smartphone detection, it achieves rapid and highly sensitive detection of nitrite in food, suitable for on-site monitoring scenarios.

[0081] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for detecting nitrite using oxygen-enriched vacancy Mn3O4 nanozymes, characterized in that: Includes the following steps: In NaAc buffer, a nitrite-containing sample is mixed with an oxygen-enriched vacancy Mn3O4 nanozyme and TOPS for reaction. Visual detection is achieved by observing the color change of the reaction system, or quantitative detection is performed by collecting the RGB values ​​of the reaction system using a smartphone and combining them with the absorbance change at 420 nm. The oxygen-enriched vacancy Mn3O4 nanozyme is prepared by the following method: Step 1: Add KMnO4 to deionized water at a ratio of 0.5-1.5g:500ml until KMnO4 is completely dissolved to obtain a mixture. Step 2: Add oleic acid to the mixture and stir continuously at 25-35°C for 4-6 hours. The volume ratio of oleic acid to deionized water in the mixture is 1:

50. Step 3: Collect the precipitate, wash it alternately with deionized water and ethanol 4 to 6 times, dry it at 70 to 90°C for 8 to 12 hours, and then calcine it at 180 to 220°C for 4 to 6 hours to obtain oxygen-rich vacancy Mn3O4 nanozyme with oxidase-like activity.

2. The method for detecting nitrite using oxygen-enriched vacancy Mn3O4 nanozymes according to claim 1, characterized in that: The NaAc buffer solution had a pH of 3.0 and a concentration of 10 mM; the oxygen-rich vacancy Mn3O4 nanozyme had a concentration of 70 μg / mL; the TOPS concentration was 200 μM; and the reaction time was 1–30 minutes.

3. The method for detecting nitrite using oxygen-enriched vacancy Mn3O4 nanozymes according to claim 2, characterized in that: In step one, the ratio of KMnO4 to deionized water is 1g:500ml, and KMnO4 is completely dissolved under stirring.

4. The application of the method as described in claim 3 in the detection of nitrite.

5. The application according to claim 4, characterized in that: The nitrites were derived from food or water samples.

6. A sensor for detecting nitrite, characterized in that: The assay includes an oxygen-rich vacancy Mn3O4 nanozyme, TOPS substrate, NaAc buffer, and a detection device. The detection device includes a visual observation module and a smartphone image analysis module. The smartphone image analysis module outputs quantitative results based on a preset RGB value-nitrite concentration calibration curve. The oxygen-rich vacancy Mn3O4 nanozyme is prepared by the following method: Step 1: Add KMnO4 to deionized water at a ratio of 0.5-1.5g:500ml until KMnO4 is completely dissolved to obtain a mixture. Step 2: Add oleic acid to the mixture and stir continuously at 25-35°C for 4-6 hours. The volume ratio of oleic acid to deionized water in the mixture is 1:

50. Step 3: Collect the precipitate, wash it alternately with deionized water and ethanol 4 to 6 times, dry it at 70 to 90°C for 8 to 12 hours, and then calcine it at 180 to 220°C for 4 to 6 hours to obtain oxygen-rich vacancy Mn3O4 nanozyme with oxidase-like activity.

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