Monatomic nano-enzyme material, preparation method thereof and application of monatomic nano-enzyme material in detection of isoniazide
By synthesizing Fe-NCP nanoenzyme materials and using their peroxidase activity, colorimetric method is established to detect isoniazid, which solves the problems of complex equipment and difficult detection in the prior art, and achieves fast, simple and accurate isoniazid detection.
Patent Information
- Application Number
- CN202510423093.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-07
- Publication Date
- 2025-07-08
Smart Images
Figure CN120268432A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of detection, and particularly relates to a single-atom nanozyme material, a preparation method thereof, and an application thereof in detecting isoniazid. Background Art
[0002] Isoniazid (INH) is a synthetic antibacterial drug. Due to its specificity and effectiveness, relatively few side effects, and low cost, it is one of the most important antibiotics for treating tuberculosis. However, unreasonable doses may not only lead to drug resistance but also cause side effects, including hepatotoxicity, neurotoxicity, and endocrine disorders. Ingesting INH at a concentration exceeding 80 mg / kg can rapidly lead to death. Therefore, it is necessary to analyze the content of INH in pharmaceutical preparations to ensure the quality of drugs and the safety of drug doses. -1
[0003] So far, various analytical methods have been developed to detect INH, including high-performance liquid chromatography, mass spectrometry, capillary electrophoresis, and electrochemistry. However, these methods often have various disadvantages, such as the need for expensive equipment, complex laboratories, and complex techniques. Colorimetric methods have received extensive attention due to their advantages such as fast response, simple operation, and clear signals. Therefore, it is of great significance to establish a simple, rapid, and accurate INH detection method based on colorimetry.
[0004] Single-metal species modified nitrogen-rich carbon (M-NC) materials are very attractive due to their unique electronic structure, high atomic utilization rate, and suitability for different catalytic applications. Catalysts with three-dimensional open structures, mesoporous or hollow structures have the potential to enhance mass transfer and increase single-atom structures. These advantages will ultimately improve the apparent activity of single-atom catalysts. The emergence of Fe-NCP reveals its unique pseudo-enzyme activity and provides potential application prospects in colorimetric sensing applications. Therefore, there is an urgent need for a comprehensive and systematic study on the rational design and engineering of Fe-NCP nanozymes, aiming to create an independent and portable detection platform for Fe-NCP. Summary of the Invention
[0005] To solve the above-mentioned existing technical problems, the present invention synthesizes Fe-NCP by a calcination method, which has a uniform spherical structure and rich active sites. After Fe doping, NCP has higher peroxidase activity, which promotes the effective catalysis of H2O2 for the oxidation of TMB. In the presence of INH, the pseudo-enzyme activity is significantly inhibited, thereby establishing a colorimetric method for INH detection.
[0006] To achieve the above object, the technical solution of the present invention is as follows: A single-atom nanozyme material, and the preparation method includes the following steps:
[0007] 1) Dissolve 2-methylimidazole and 5-aminotetrazole in methanol to form solution A, dissolve zinc acetate in methanol to form solution B, pour solution A into solution B and stir, let the resulting suspension stand, centrifuge, wash with ethanol, and dry at 70 °C to obtain an intermediate product;
[0008] 2) Place the intermediate product obtained in step 1) in a porcelain boat, then put it into a tubular furnace and calcine under nitrogen protection to obtain a solid product; soak the obtained solid product in a hydrochloric acid solution, wash with deionized water, centrifuge, and dry at 70 °C to obtain intermediate NCP;
[0009] 3) Dissolve ferric chloride and the intermediate NCP obtained in step 2) in ethanol, mix evenly, add ammonium chloride after drying, mix and grind evenly, and then calcine the resulting mixture under a nitrogen atmosphere to obtain a single-atom nanozyme material Fe-NCP.
[0010] For the above single-atom nanozyme material, in step 1), by mass ratio, 2-methylimidazole:5-aminotetrazole:zinc acetate = 246:77:214.
[0011] For the above single-atom nanozyme material, in step 1), the standing is carried out at 60 °C for 100 min.
[0012] For the above single-atom nanozyme material, in step 2), the calcination temperature is 700 °C for 2 h, and the heating rate is 5 °C . min -1 。
[0013] For the above single-atom nanozyme material, in step 2), the hydrochloric acid concentration is 1 mol / L, and the soaking time is 12 h.
[0014] For the above single-atom nanozyme material, in step 3), by mass ratio, Fe 3+ :NCP = 1:50.
[0015] For the above single-atom nanozyme material, in step 3), the calcination temperature is 500 °C for 1 h, and the heating rate is 5 °C . min -1 。
[0016] Application of the above single-atom nanozyme material in detecting isoniazid.
[0017] For the above application, the method is as follows: Add water to the above single-atom nanozyme material to form an aqueous solution, then drop an isoniazid solution into it, and detect the concentration of isoniazid by colorimetry according to the absorbance value.
[0018] For the above application, the concentration of the single-atom nanozyme material is 0.1 mg·ml-1 , pH = 3.5, and the temperature is 35 °C.
[0019] The beneficial effects of the present invention are as follows:
[0020] The single-atom nanozyme material described in the present invention has abundant active sites and excellent peroxidase-mimicking activity. This material has a low detection limit for the detection of isoniazid. The detection method described in the present invention is convenient and fast, which is conducive to detection. Description of the Drawings
[0021] Figure 1 is the SEM image of Fe-NCP.
[0022] Figure 2 is the comparison chart of the enzymatic activities of Fe-NCP and NCP materials.
[0023] Figure 3 is the optimization chart of the conditions of Fe-NCP. pH (A), temperature (B).
[0024] Figure 4 is the linear relationship chart of Fe-NCP and IHN.
[0025] Figure 5 is the (a) selectivity experiment and (b) anti-interference experiment for the detection of INH by Fe-NCP. Detailed Embodiments
[0026] Example 1 Preparation of a Single-Atom Nanozyme Material
[0027] (I) The preparation method is as follows
[0028] 1. Preparation of ZTF-AP
[0029] Dissolve 246 mg of 2-methylimidazole and 77 mg of 5-aminotetrazole in 10 ml of methanol to form solution A. Dissolve 214 mg of zinc acetate in 10 ml of methanol to form solution B. Pour solution A into solution B and stir for 5 min. Let the obtained suspension stand at 60 °C for 100 min, centrifuge, wash with ethanol, and dry at 70 °C to obtain an intermediate product;
[0030] 2. Preparation of NCP
[0031] Place the intermediate product obtained in step 1 in a porcelain boat, then put it into a tubular furnace, and calcine at 700 °C for 2 h under nitrogen protection to obtain a solid product; soak the obtained solid product in 1 M hydrochloric acid solution for 12 h, wash with deionized water, centrifuge, and dry at 70 °C to obtain the intermediate NCP;
[0032] 3. Preparation of Fe-NCP
[0033] In 50 μL of FeCl3. In a 6H2O ethanol solution (containing 0.2 mg Fe 3+ ), 10 mg of NCP dissolved in ethanol was added, mixed evenly, dried, 100 mg of ammonium chloride was added, mixed and ground evenly, and then calcined at 500 °C for 1 h under nitrogen protection to obtain the single-atom nanozyme Fe-NCP.
[0034] (II) Detection
[0035] 1. SEM was used to study and analyze the morphology of Fe-NCP.
[0036] Figure 1 is the scanning electron microscope image of the prepared Fe-NCP, which is a spherical structure.
[0037] 2. Evaluation of the peroxidase activity of Fe-NCP.
[0038] To verify the peroxidase activity of Fe-NCP, we added 50 μL of 5 mM TMB solution, 50 μL of 15% H2O2 solution, and 870 μL of acetate buffer solution to 30 μL of Fe-NCP with a concentration of 0.1 mg / ml, incubated at 35 °C for 20 min, and measured the ultraviolet absorbance at 652 nm. Figure 2 Only when TMB and H2O2 coexist, Fe-NCP catalyzes the oxidation of colorless TMB to blue oxTMB, and the absorbance center is located at 652 nm, indicating that Fe-NCP exhibits amazing peroxidase catalytic activity. At 652 nm, the absorbance of the H2O2-TMB system is relatively low. In contrast, after adding Fe-NCP, significant changes with ultraviolet-visible absorption peak characteristics appear in the solution, indicating that Fe-NCP has excellent peroxidase activity and can catalyze the oxidation of TMB induced by H2O2.
[0039] 2. Optimization of the peroxidase activity of Fe-NCP.
[0040] As Figure 3 , Figure 3 in (A), the pH of the buffer solution was optimized. We added 50 μL of 5 mM TMB solution, 50 μL of 15% H2O2 solution, and 870 μL of acetate buffer solution with different pH values (3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8) to 30 μL of Fe-NC with a concentration of 0.1 mg / ml, incubated at 35 °C for 20 min, and measured the ultraviolet absorbance at 652 nm. It was found that when the pH of the buffer solution was 3.5, the absorbance was the highest, proving that the peroxidase activity of Fe-NCP was the highest at this pH value. Therefore, the optimal pH was 3.5. Similarly, Figure 3In (B), the temperature was optimized. Only the incubation temperature was changed, and it was found that the absorbance was the highest at 35 °C, proving that the peroxidase activity of Fe-NCP was the highest at this temperature, and the optimum temperature was 35 °C.
[0041] Example 2 Detection of isoniazid using Fe-NCP (I) Effect of different concentrations of isoniazid on absorbance
[0042] Using Fe-NCP with a concentration of 0.1 mg·ml -1 Isoniazid was detected under the conditions of pH = 3.5 and a temperature of 35 °C. 30 μL of isoniazid solutions with different concentrations were added to 30 μL of Fe-NCP and 840 μL of buffer solution, incubated for 15 min, then 50 μL of TMB (5 mM) and 50 μL of H2O2 (15%) were added, incubated for 20 min, and the absorbance was measured at a wavelength of 652 nm. As Figure 4 As the concentration of isoniazid increased, the absorbance signal decreased. And a linear relationship was presented between the absorbance and the concentration.
[0043] (II) Selectivity and anti-interference experiments for the detection of isoniazid using Fe-NCP
[0044] 1. Selectivity experiment for the detection of isoniazid using Fe-NCP
[0045] Figure 5 In (a), it was the selectivity experiment for the detection of isoniazid by Fe-NCP. We added 50 μL of TMB solution, 50 μL of H2O2 solution, 30 μL of Fe-NCP dispersion, and 30 μL of 1.5 mmol L -1 interference ions (Na + , K + , Ca 2+ , Zn 2+ , Cl - , NO3 - , SO4 2- ) to 840 μL of acetate buffer solution, mixed well and reacted at 35 °C for 20 minutes, and the maximum absorbance of the reaction solution at 652 nm was measured using a UV-visible absorption spectrometer. As Figure 5 As shown in (a), most of the interfering substances were not significant, indicating good selectivity for isoniazid.
[0046] 2. Anti-interference experiment for the detection of isoniazid using Fe-NCP
[0047] Figure 5Figure (b) shows the anti-interference experiment of Fe-NCP for isoniazid detection. We added 50 μL of TMB solution, 50 μL of H2O2 solution, 30 μL of Fe-NCP dispersion, 30 μL of INH solution, and 30 μL of 1.5 mmol / L -1 interference ions (Na + , K + , Ca 2+ , Zn 2+ , Cl - , NO3 - , SO4 2- ) to 810 μL of acetate buffer solution, mixed well, and reacted at 35 °C for 20 minutes. The maximum absorbance of the reaction solution at 652 nm was measured using a UV-visible absorption spectrometer. As Figure 5 shown in (b), when the above substances are present in the reaction system containing Fe-NCP, TMB, and H2O2, the UV absorption intensity is approximately equal to the absorption intensity containing only INH. The results show that the detection system has good anti-interference ability for INH activity.
[0048] Evaluation of the detection effect of isoniazid concentration in actual samples in Example 3
[0049] During the detection process, urine and blood were used as actual samples to analyze isoniazid. Different concentrations of analytes (2.4 μmol / L -1 , 9 μmol / L -1 , 18 μmol / L -1 ) were added to the samples. The precision and accuracy of the method were studied by the standard addition method.
[0050] Table 1
[0051]
[0052] As can be seen from Table 1, Fe-NCP has excellent analytical performance for isoniazid, and the spiked recovery rates are between 85.98% and 101.11%. It shows that the sensor has a high recovery rate and good precision, and can be used for the detection of trace isoniazid in actual samples.
Claims
1. A single-atom nanozyme material, characterized in that, The preparation method includes the following steps: 1) Dissolve 2-methylimidazole and 5-aminotetrazole in methanol to form solution A, dissolve zinc acetate in methanol to form solution B, pour solution A into solution B and stir, let the obtained suspension stand, centrifuge, wash with ethanol, and dry at 70 °C to obtain an intermediate product; 2) Place the intermediate product obtained in step 1) in a porcelain boat, then put it into a tube furnace and calcine under nitrogen protection to obtain a solid product; soak the obtained solid product in a hydrochloric acid solution, wash with deionized water, centrifuge, and dry at 70 °C to obtain intermediate NCP; 3) Dissolve ferric chloride and the intermediate NCP obtained in step 2) in ethanol, mix evenly, add ammonium chloride after drying, mix and grind evenly, and then calcine the obtained mixture under a nitrogen atmosphere to obtain the single-atom nanozyme material Fe-NCP.
2. The single-atom nanozyme material according to claim 1, wherein In step 1), by mass ratio, 2-methylimidazole:5-aminotetrazole:zinc acetate = 246:77:
214.
3. The single-atom nanozyme material according to claim 1, characterized in that In step 1), the standing is carried out at 60 °C for 100 min.
4. The single-atom nanozyme material according to claim 1, characterized in that In step 2), the calcination temperature is 700 °C for 2 h, and the heating rate is 5 °C . min -1 .
5. A single-atom nanozyme material according to claim 1, characterized in that In step 2), the concentration of the hydrochloric acid is 1 mol / L, and the soaking time is 12 h.
6. The single-atom nanozyme material according to claim 1, wherein In step 3), by mass ratio, Fe 3 + : NCP = 1:
50.
7. The single-atom nanozyme material according to claim 1, wherein In step 3), the calcination temperature is 500 °C for 1 h, and the heating rate is 5 °C . min -1 .
8. Use of a single-atom nanozyme material according to any one of claims 1-7 in the detection of isoniazid.
9. The application according to claim 8, wherein The method is as follows: Add water to the single-atom nanozyme material described in claim 1 to form an aqueous solution, then drop an isoniazid solution into it, and detect the concentration of isoniazid by colorimetry according to the absorbance value.
10. The application according to claim 9, characterized in that, The concentration of the single-atom nanozyme material is 0.1 mg·ml -1 , pH = 3.5, and the temperature is 35°C.