Preparation method of octahedral Ag2O nano-enzyme and method for detecting H2O2 by colorimetric method
By preparing octahedral Ag2O nanozymes as catalysts, and combining colorimetry and ultraviolet spectrophotometry, the problems of complexity and high cost of existing H2O2 detection methods have been solved, achieving simple, rapid and accurate H2O2 detection, which is applicable to the food and chemical industries.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XIAN INT UNIV
- Filing Date
- 2025-12-17
- Publication Date
- 2026-05-05
AI Technical Summary
Existing H2O2 detection methods are complex, costly, and require highly skilled operators, lacking simple, rapid, and accurate detection methods.
An octahedral Ag2O nanozyme was used as a catalyst. H2O2 was detected by colorimetry. The morphology and size of the nanozyme were characterized by X-ray diffraction and scanning electron microscopy. The absorbance of the reaction system was measured by ultraviolet spectrophotometer. A simple and rapid method for H2O2 detection was established.
It enables the visual detection of H2O2, exhibiting good selectivity and accuracy. It is suitable for the detection of H2O2 in real samples, with high recovery rate and small relative standard deviation, making it applicable to the food and chemical industries.
Smart Images
Figure CN121972160A_ABST
Abstract
Description
Technical Field
[0001] This disclosure relates to the field of analyte concentration detection technology, specifically to a method for preparing octahedral Ag2O nanozymes and a colorimetric method for detecting H2O2. Background Technology
[0002] H2O2 is a green, common, and high-quality chemical reagent widely used in chemical, food, and hygiene fields, and has become a popular choice in the industry. H2O2 can serve as a biomarker for serious diseases and can also be used as a food additive. Excessive concentrations of H2O2 entering the human body can generate excessive hydroxyl radicals, posing serious health risks. Other methods for detecting H2O2 include chemical titration and electrochemical methods, but these methods require more specialized operators, stricter experimental environments, and more expensive equipment. Compared to these methods, this disclosure presents a colorimetric detection method for H2O2 based on nanozymes and peroxidase-like activity, which is more visual, faster, and less costly. Based on the experimental subjects of this disclosure, it can contribute to the search for novel nanozymes to construct colorimetric sensors to improve detection sensitivity, and is of great significance for developing a simpler, faster, and more accurate detection method for H2O2. Summary of the Invention
[0003] The colorimetric method for detecting H2O2 disclosed herein has the advantages of simple operation, accurate detection, and visualized results.
[0004] According to a first aspect of the present disclosure, a method for preparing octahedral Ag2O nanozymes is provided, the method comprising the following steps:
[0005] Step S1: Prepare nitric acid solution, ammonia solution and sodium hydroxide solution;
[0006] Step S2: Slowly add the ammonia solution to the nitric acid solution to form a silver ammonia solution and stir for a preset time. While stirring, add the sodium hydroxide solution to the mixture dropwise. After a large amount of brown precipitate is formed, leave the precipitate in the solution for a preset time, collect and wash the precipitate to obtain octahedral Ag2O nanozyme powder.
[0007] Step S3: Characterize the Ag2O nanozyme powder using X-ray diffraction;
[0008] Step S4: Characterize the morphology and size of the Ag2O nanozyme powder using scanning electron microscopy.
[0009] In one embodiment, in step S1
[0010] The preparation of silver nitrate solution includes: weighing 0.8494 g of silver nitrate into a beaker and adding 45 mL of pure water and stirring until fully dissolved. Then, transfer the silver nitrate solution to a 50 mL volumetric flask, make up to volume with a dropper, and label the flask to obtain a silver nitrate solution with a concentration of 0.1 mol / L.
[0011] The preparation of an ammonia solution involves: taking 376 μL of 25% ammonia solution, adding sufficient pure water to dilute it into a 50 mL volumetric flask, and obtaining an ammonia solution with a concentration of 0.1 mol / L;
[0012] The preparation of sodium hydroxide solution includes: weighing 2.000 g of sodium hydroxide into a beaker and adding 20 mL of pure water, stirring continuously until the sodium hydroxide is completely dissolved. Then, transfer the sodium hydroxide solution to a 25 mL volumetric flask, dilute to volume with a dropper, and label the flask to obtain a sodium hydroxide solution with a concentration of 2.0 mol / L.
[0013] In one embodiment, in step S2
[0014] 50 mL of 0.1 mol / L AgNO3 solution was placed in a beaker. Under constant temperature and magnetic stirring at 600 r / min, 25 mL of 0.1 mol / L ammonia water was slowly added dropwise to form a silver ammonia solution. After stirring for 10 min, 2.5 mL of 2 mol / L NaOH solution was added dropwise to the mixture while stirring. After a large amount of brown precipitate was formed, the precipitate was left in the solution for 12 h. The precipitate was then centrifuged at 8000 rpm for 25 min. The precipitate was collected and washed with ethanol and pure water until the pH of the final aqueous solution was neutral, yielding octahedral Ag2O nanozyme powder.
[0015] In one embodiment, in step S3
[0016] The octahedral Ag2O nanozyme powder was dispersed in a square frosted area of a glass slide and spread evenly. After being flattened with a coverslip, it was characterized by X-ray diffraction.
[0017] In one embodiment, in step S4
[0018] The conductive adhesive was fixed on the sample stage. The octahedral Ag2O nanozyme powder was ground into powder and then dotted onto the conductive adhesive. After being blown and spread evenly, it was placed in a scanning electron microscope for inspection and photographs were taken.
[0019] According to a second aspect of the present disclosure, a method for colorimetric detection of H2O2 is provided, the method comprising the following steps:
[0020] Step S11: Take several centrifuge tubes and add a measured amount of A to each centrifuge tube. g2O solution and H2O2 of different concentrations are mixed evenly; wherein, the A g2 Solution O is based on the octahedral A described above. g2 It was prepared using the method for preparing O nanozymes;
[0021] Step S12: After adding NaAc-Hac buffer and TMB solution to each centrifuge tube processed in step S11, an Ag2O-TMB-H2O2 reaction system is formed. After heating the Ag2O-TMB-H2O2 reaction system in each centrifuge tube in a water bath for a preset time, the color change of the solution in each centrifuge tube is observed.
[0022] Step S13: Measure the absorbance of the Ag2O-TMB-H2O2 reaction system at a preset wavelength using an ultraviolet spectrophotometer and record the data.
[0023] In one embodiment, prior to step S12, the method further includes: preparing a NaAc-Hac buffer solution and a TMB solution; wherein,
[0024] The preparation of NaAc-Hac buffer solution includes: weighing 4.1017 g of anhydrous sodium acetate into a container, adding 2.86 mL of glacial acetic acid, and then adding sufficient pure water to prepare 500 mL of 0.1 mol / L buffer solution;
[0025] The preparation of TMB solution involves: weighing 0.2500 g of 3,3′,5,5′-tetramethylbenzidine into a container, then adding 1.5 mL of anhydrous ethanol, heating and stirring constantly to completely dissolve it, and then making up to 25 mL in a volumetric flask to obtain a 41.6 mmol / L TMB solution.
[0026] In one embodiment, the method includes:
[0027] In step S11: Take several 5 mL centrifuge tubes and add 50 μL of A to each centrifuge tube. g2 O solution and 20 μL of H2O2 of different concentrations are mixed evenly;
[0028] In step S12: 3810 μL of 0.1 mol / L NaAc-Hac buffer solution with a pH of 4.5 and 120 μL of TMB solution were added to each centrifuge tube after the treatment in step S11 to form an Ag2O-TMB-H2O2 reaction system. After heating the Ag2O-TMB-H2O2 reaction system in each centrifuge tube in a water bath at 25 ℃ for 50 min, the color change of the solution in each centrifuge tube was observed with the naked eye.
[0029] In step S13, the absorbance of the Ag2O-TMB-H2O2 reaction system at a wavelength of 652 nm is measured using a T2600 UV spectrophotometer and the data is recorded.
[0030] In one embodiment, the method further includes the following steps:
[0031] Step S14: Measure 5 mL of the test solution into a 50 mL beaker using a graduated cylinder, add 45 mL of 0.1 mol / L NaAc-HAc buffer solution and stir to mix well. After standing for 15 min, centrifuge at 8000 r / min for 20 min and take the supernatant as the solvent to prepare the spiked sample of H2O2.
[0032] Step S15: Add the spiked sample to a NaAc-HAc buffer solution containing Ag2O nanozyme and TMB solution to form a reaction solution; wherein, in the reaction solution, the concentration of Ag2O nanozyme is 125 μmol / L, the concentration of TMB is 0.225 mmol / L, and the concentrations of H2O2 in the spiked sample are 5 μmol / L, 10 μmol / L, and 50 μmol / L, respectively;
[0033] In step S16, after heating the solution treated in step S15 in a water bath at 25 ℃ for 50 min, the absorption spectrum of the reaction solution at 652 nm was measured using a T2600 UV spectrophotometer and the data was recorded. The recovery rate was then calculated.
[0034] This disclosure provides a colorimetric method for detecting H2O2. When investigating the influence of potential interfering substances on the H2O2 detection system, the concentration of H2O2 is selected as 10 μmol / L, and K... + Pb 2+ Cu 2+ Ni 2+ and Fe 2+ Both were 100 μmol / L, used to determine H2O2 and K. + Pb 2+ Cu 2+ Ni 2+ and Fe 2+ The effect of the single substance on the absorbance value of the measurement system was investigated, and the standard deviation was found to be less than 10%, and ten times that of K. + Pb 2+ Cu 2+ Ni 2+ and Fe 2+ It does not interfere with the detection of H2O2, thus it can be seen that the colorimetric method for detecting H2O2 disclosed herein has good selectivity for H2O2 detection. Attached Figure Description
[0035] Figure 1The diagram shows the detailed preparation steps of the octahedral Ag2O in this embodiment.
[0036] Figure 2 A flowchart of a colorimetric method for detecting H2O2 provided in an embodiment of this disclosure.
[0037] Figure 3 This is a diagram illustrating the reaction mechanism of Ag2O nanozyme catalyzing TMB in the embodiments of this disclosure.
[0038] Figure 4 This is a schematic diagram illustrating the principle of visual colorimetric detection of H2O2 using Ag2O nanozymes in this embodiment of the present disclosure.
[0039] Figure 5 The XRD phase characterization pattern of Ag2O powder in the embodiments of this disclosure is shown.
[0040] Figure 6 This is a SEM image of the Ag2O nanomaterial in the embodiments of this disclosure.
[0041] Figure 7 This is the ultraviolet-visible spectrum of the reaction system in the embodiments of this disclosure for feasibility testing.
[0042] Figure 8 This is a schematic diagram illustrating the effect of solution pH on the catalytic activity of nanozymes in the embodiments of this disclosure.
[0043] Figure 9 This is a schematic diagram illustrating the effect of temperature on absorbance in the reaction system of this embodiment.
[0044] Figure 10 This is a schematic diagram illustrating the effect of different TMB concentrations on the absorbance of the reaction system in the embodiments of this disclosure.
[0045] Figure 11 This is a schematic diagram illustrating the effect of different Ag2O concentrations on the absorbance value of the reaction system in the embodiments of this disclosure.
[0046] Figure 12 This is a schematic diagram illustrating the effect of different reaction times on the absorbance value of the reaction system in the embodiments of this disclosure.
[0047] Figure 13 This is the ultraviolet absorption spectrum of the Ag2O-H2O2-TMB detection system in the embodiments of this disclosure.
[0048] Figure 14 According to Figure 13 The linear fit plot of the detection system in this embodiment is drawn from the absorbance values in the ultraviolet spectrum.
[0049] Figure 15 This is a schematic diagram illustrating the effect of interfering ions on the detection system in an embodiment of this disclosure. Detailed Implementation
[0050] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of systems consistent with some aspects of this disclosure as detailed in the appended claims.
[0051] This disclosure provides a method for preparing an octahedral Ag2O nanozyme, which includes the following steps:
[0052] Step S1: Prepare nitric acid solution, ammonia solution and sodium hydroxide solution;
[0053] In one embodiment, in step S1
[0054] The preparation of silver nitrate solution includes: weighing 0.8494 g of silver nitrate into a beaker and adding 45 mL of pure water, stirring until fully dissolved, transferring the silver nitrate solution to a 50 mL volumetric flask, and making up to volume with a dropper before labeling to obtain a silver nitrate solution with a concentration of 0.1 mol / L; it should be noted that the silver nitrate solution in this embodiment needs to be stored away from light.
[0055] The preparation of the ammonia solution involves: taking 376 μL of 25% ammonia solution, adding sufficient pure water to dilute it into a 50 mL volumetric flask to obtain an ammonia solution with a concentration of 0.1 mol / L. It should be noted that in this embodiment, in order to keep the ammonia solution concentration constant, the ammonia solution should be kept away from light and stored as soon as possible.
[0056] The preparation of sodium hydroxide solution includes: weighing 2.000 g of sodium hydroxide into a beaker and adding 20 mL of pure water, stirring continuously until the sodium hydroxide is completely dissolved. Then, transfer the sodium hydroxide solution to a 25 mL volumetric flask, dilute to volume with a dropper, and label the flask to obtain a sodium hydroxide solution with a concentration of 2.0 mol / L.
[0057] Step S2: Slowly add the ammonia solution to the nitric acid solution to form a silver ammonia solution and stir for a preset time. While stirring, add the sodium hydroxide solution to the mixture dropwise. After a large amount of brown precipitate is formed, leave the precipitate in the solution for a preset time, collect and wash the precipitate to obtain octahedral Ag2O nanozyme powder.
[0058] In one embodiment, in step S2, 50 mL of 0.1 mol / L AgNO3 solution is placed in a beaker. Under constant temperature and magnetic stirring at 600 r / min, 25 mL of 0.1 mol / L ammonia water is slowly added dropwise to form a silver ammonia solution. After stirring for 10 min, 2.5 mL of 2 mol / L NaOH solution is added dropwise to the mixture while stirring. After a large amount of brown precipitate is formed, the precipitate is left in the solution for 12 h. The precipitate is then centrifuged at 8000 rpm for 25 min. The precipitate is collected and washed with ethanol and pure water until the pH of the final aqueous solution is neutral to obtain octahedral Ag2O nanozyme powder.
[0059] In this embodiment, an ammonia-sodium hydroxide stepwise precipitation method is used to prepare octahedral Ag₂O nanozyme powder. Specifically, silver nitrate solution is first mixed with ammonia to generate silver hydroxide precipitate, and then the silver hydroxide precipitate is centrifuged to obtain octahedral Ag₂O nanozyme powder.
[0060] In this embodiment, octahedral Ag₂O nanomaterials were prepared using the ammonia method with silver nitrate and ammonia as raw materials. First, 0.8494 g of silver nitrate powder was dissolved in water to prepare 50 mL of 0.1 mol / L AgNO₃ solution. Then, 50 mL of the 0.1 mol / L AgNO₃ solution was placed in a beaker. Under constant temperature and magnetic stirring at 600 rpm, 25 mL of 0.1 mol / L ammonia was slowly added dropwise, forming a silver ammonia solution. After stirring for 10 min, 2.5 mL of 2 mol / L NaOH was added dropwise while stirring. When a large amount of brown precipitate formed, the precipitate was left in the solution for 12 h, and the nanomaterials were centrifuged at 8000 rpm for 25 min. The precipitate was collected and washed with ethanol and pure water until the pH of the final aqueous solution was neutral. Finally, the obtained octahedral Ag₂O was dried at 60 °C and stored in the dark.
[0061] Figure 1 The diagram shows the detailed preparation steps of the octahedral Ag2O in this embodiment.
[0062] Step S3: Characterize the Ag2O nanozyme powder using X-ray diffraction;
[0063] In one embodiment, octahedral Ag2O nanozyme powder is dispersed in a square frosted area of a glass slide and spread evenly. After being flattened with a coverslip, it is characterized by X-ray diffraction.
[0064] In this embodiment, the prepared Ag2O powder was characterized using X-ray diffraction (XRD). Specifically, Ag2O powder was evenly spread in a square frosted area of a specially made glass slide, and then flattened with a coverslip, keeping the sample flush with the edge of the groove on the glass slide.
[0065] Step S4: Characterize the morphology and size of the Ag2O nanozyme powder using scanning electron microscopy.
[0066] In one embodiment, in step S4, the conductive adhesive is fixed on the sample stage, the octahedral Ag2O nanozyme powder is ground into powder and then dotted onto the conductive adhesive. After being blown and spread evenly, it is placed in a scanning electron microscope for inspection and photographs are taken.
[0067] In this embodiment, the morphology and size of the prepared Ag2O nanomaterials were characterized using a scanning electron microscope. Specifically, the conductive adhesive was fixed on the sample stage, the nanomaterials were ground, and an appropriate amount of powder was dotted onto the conductive adhesive. After being evenly spread by blowing with a syringe, the sample was placed in the instrument for detection, and photographs were taken.
[0068] In this embodiment, the method for preparing octahedral Ag₂O nanozymes employs a chemical method suitable for convenient laboratory preparation to produce octahedral Ag₂O nanoparticles. However, existing Ag₂O nanoparticles are synthesized through chemical precipitation of silver ions with strong bases such as sodium hydroxide. While this method offers rapid synthesis, the size and morphology of Ag₂O are difficult to control. Therefore, this embodiment adjusts the synthesis rate of Ag₂O nanoparticles by adding ammonia. Specifically, when ammonia is slowly added dropwise to an AgNO₃ solution and stirred for 10 minutes, Ag₂O and NH₄NO₃ first form an intermediate [Ag(NH)₂], thereby reducing the chemical reaction rate between sodium hydroxide and Ag₂O. Furthermore, to synthesize Ag₂O nanocrystals with different morphologies, this embodiment modifies the molar ratio of Ag₂O / NH₄NO₃ to NaOH. Moreover, to obtain uniformly shaped nanocrystals during the preparation process, this embodiment uses a disposable IV drip set to control the dripping rate of different solutions.
[0069] Figure 2 A flowchart illustrating a colorimetric method for detecting H2O2 provided in this disclosure. Figure 2 As shown, the method includes:
[0070] Step S11: Take several centrifuge tubes and add a measured amount of A to each centrifuge tube. g2 O solution and H2O2 of different concentrations are mixed evenly; wherein, the A g2 Solution O is the octahedral A described above. g2 It was prepared using the method for preparing O nanozymes;
[0071] In one embodiment, several 5 mL centrifuge tubes are taken, and 50 μL of A is added to each centrifuge tube. g2 The O solution and 20 μL of H2O2 of different concentrations were mixed evenly;
[0072] Step S12: After adding NaAc-Hac buffer and TMB solution to each centrifuge tube processed in step S11, an Ag2O-TMB-H2O2 reaction system is formed. After heating the Ag2O-TMB-H2O2 reaction system in each centrifuge tube in a water bath for a preset time, the color change of the solution in each centrifuge tube is observed.
[0073] In one embodiment, after adding 3810 μL of 0.1 mol / L NaAc-Hac buffer solution with a pH of 4.5 and 120 μL of TMB solution to each centrifuge tube after step S11, an Ag2O-TMB-H2O2 reaction system is formed. After heating the Ag2O-TMB-H2O2 reaction system in each centrifuge tube in a water bath at 25 °C for 50 min, the color change of the solution in each centrifuge tube is observed visually.
[0074] Step S13: Measure the absorbance of the Ag2O-TMB-H2O2 reaction system at a preset wavelength using an ultraviolet spectrophotometer and record the data.
[0075] In one embodiment, the absorbance of the Ag2O-TMB-H2O2 reaction system at a wavelength of 652 nm was measured and the data was recorded using a T2600 UV spectrophotometer, with each sample measured at least three times in parallel.
[0076] Optionally, before performing step S12, the method further includes: preparing NaAc-Hac buffer and TMB solution; wherein,
[0077] To prepare the NaAc-Hac buffer solution, weigh 4.1017 g of anhydrous sodium acetate into a container, add 2.86 mL of glacial acetic acid, and then add sufficient pure water to prepare a 500 mL 0.1 mol / L buffer solution.
[0078] The preparation of TMB solution involves: weighing 0.2500 g of 3,3′,5,5′-tetramethylbenzidine into a container, then adding 1.5 mL of anhydrous ethanol, heating and stirring constantly to completely dissolve it, and then making up to 25 mL in a volumetric flask to obtain a 41.6 mmol / L TMB solution.
[0079] Optionally, the method further includes the following steps:
[0080] Step S14: Measure 5 mL of the test solution into a 50 mL beaker using a graduated cylinder, add 45 mL of 0.1 mol / L NaAc-HAc buffer solution and stir to mix well. After standing for 15 min, centrifuge at 8000 r / min for 20 min and take the supernatant as the solvent to prepare the spiked sample of H2O2.
[0081] Step S15: Add the spiked sample to a NaAc-HAc buffer solution containing Ag2O nanozyme and TMB solution to form a reaction solution; wherein, in the reaction solution, the concentration of Ag2O nanozyme is 125 μmol / L, the concentration of TMB is 0.225 mmol / L, and the concentrations of H2O2 in the spiked sample are 5 μmol / L, 10 μmol / L, and 50 μmol / L, respectively;
[0082] In step S16, after heating the solution treated in step S15 in a water bath at 25 ℃ for 50 min, the absorption spectrum of the reaction solution at 652 nm was measured using a T2600 UV spectrophotometer and the data was recorded. The recovery rate was then calculated.
[0083] For example, in order to verify the detection effect of the sensing system on H2O2 in actual samples, this disclosure uses milk as the test sample, which is purchased from the college supermarket and the actual sample is pre-processed. Specifically, 5 mL of milk was measured using a graduated cylinder and placed in a 50 mL beaker. Then, 45 mL of 0.1 mol / L NaAc-HAc buffer (pH 4.5) was added, stirred and mixed, and allowed to stand for 15 min. After centrifugation at 8000 r / min for 20 min, the supernatant was used as a solvent to prepare a spiked sample of H2O2. The spiked sample was then added to a NaAc-HAc buffer (pH 4.5) containing Ag2O nanomaterials and TMB solution. The final reaction solution contained Ag2O nanomaterials and TMB at concentrations of 125 μmol / L and 0.225 mmol / L, respectively, and H2O2 at concentrations of 5 μmol / L, 10 μmol / L, and 50 μmol / L, respectively. After heating in a water bath at 25 ℃ for 50 min, the absorption spectrum of the reaction solution at 652 nm was measured using a T2600 UV spectrophotometer and the data was recorded. The recovery rate was calculated, and each sample was measured in at least three parallel determinations.
[0084] Figure 3 This is a diagram illustrating the reaction mechanism of Ag2O nanozyme catalyzing TMB in the embodiments of this disclosure.
[0085] Figure 4 This is a schematic diagram illustrating the principle of visual colorimetric detection of H2O2 using Ag2O nanozymes in this embodiment of the present disclosure.
[0086] like Figure 3 and Figure 4As shown, Ag₂O nanomaterials, with their peroxidase-like activity, catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂, transforming it from colorless to a blue oxidation product (oxTMB). This system can be detected by ultraviolet spectrophotometer and the color change can also be observed with the naked eye, achieving visualization. By using Ag₂O nanozymes to catalyze the oxidation of TMB by H₂O₂, a rapid and accurate colorimetric sensor for detecting H₂O₂ is established.
[0087] Figure 5 The image shows the XRD phase characterization pattern of Ag2O powder in the embodiments of this disclosure. Figure 5 As shown in the present embodiment, the Ag2O nanoparticles prepared by the ammonia method were characterized by X-ray diffraction. Characteristic diffraction peaks appeared at 2θ of 32.790°, 38.066°, 54.903°, 65.442° and 68.754°, corresponding to crystal planes (111), (200), (220), (311) and (222), which are basically the same as the data disclosed in the prior art.
[0088] Figure 6 This is a SEM image of the Ag2O nanomaterial in an embodiment of this disclosure. Figure 6 As shown in the embodiments of this disclosure, scanning electron microscopy (SEM) is used to characterize the morphology and structure of Ag2O nanoparticles prepared by the ammonia method.
[0089] Figure 7 This is the UV-Vis spectrum of the reaction system used in the feasibility test of this embodiment. This embodiment investigates whether Ag2O nanozymes possess peroxidase-like activity in the catalytic oxidation of TMB by H2O2 using Ag2O nanozymes as a catalyst. Figure 7 As shown, in the TMB-H2O2 reaction system, the solution color hardly changed; in the TMB-Ag2O reaction system, a light blue color was observed to the naked eye. When Ag2O nanoparticles were added to the TMB-H2O2 reaction system, the rate of H2O2 oxidation of TMB significantly increased, and the solution changed from colorless to blue. Furthermore, among the three systems, the Ag2O-H2O2-TMB system exhibited the highest absorption peak intensity at 652 nm, indicating that the Ag2O nanozyme has high catalytic activity; secondly, the TMB-Ag2O reaction system had the highest absorption peak intensity, while the TMB-H2O2 reaction system had the lowest. Therefore, the detection system in this embodiment can catalyze a blue color change reaction of TMB in the presence of H2O2, achieving visualized detection of H2O2. It should be noted that... Figure 7In the diagram, a represents the TMB-H₂O₂ reaction system; b represents the TMB-Ag₂O reaction system; and c represents the Ag₂O-H₂O₂-TMB reaction system, where TMB is 0.225 mmol / L. g2 O is 125 μmol / L; H2O2 is 500 μmol / L.
[0090] The embodiments disclosed herein also optimize the pH value of the buffer solution, the reaction temperature, the TMB concentration, the Ag2O concentration, and the system reaction time, which will be described in detail below.
[0091] Figure 8 This diagram illustrates the effect of solution pH on the catalytic activity of nanozymes in embodiments of this disclosure. Figure 8 As shown, in this embodiment of the disclosure, the absorbance changes at different buffer pH values (3.0, 3.5, 4.0, 4.5, and 5.0) were compared in the experiment. Figure 8 It can be seen that as the pH of the buffer solution increases from 3.0 to 5.0, the absorbance of the reaction system at 652 nm shows a trend of first increasing and then decreasing. When the pH of the Ag2O-H2O2-TMB system is 4.5, the absorbance of the reaction system reaches its maximum value, and the nanozyme catalytic activity is the highest. Based on this, it can be concluded that using a buffer solution with a pH of 4.5 as the reaction system condition in the embodiments of this disclosure can obtain the best results.
[0092] Figure 9 This diagram illustrates the effect of temperature on absorbance in the reaction system according to embodiments of this disclosure. Figure 9 As shown, embodiments of this disclosure compare the changes in absorbance values at different reaction temperatures (20 °C, 25 °C, 30 °C, 35 °C, and 40 °C). According to... Figure 9 It can be seen that as the reaction temperature increases, the absorbance value of the reaction system at 652 nm first increases and then decreases. Subsequently, as the reaction temperature continues to rise, the absorbance of the reaction system gradually weakens, reaching its maximum value at 25 °C. Based on this, it can be concluded that using a reaction temperature of 25 °C as the reaction system condition in this embodiment of the present disclosure can achieve the best results.
[0093] Figure 10 This diagram illustrates the effect of different TMB concentrations on the absorbance of the reaction system in embodiments of this disclosure. Figure 10 As shown, in the embodiments of this disclosure, the TMB concentration is between 0.075 mmol / L and 0.375 mmol / L. According to... Figure 10It can be seen that as the TMB concentration gradually increases, the absorbance of the reaction system at 652 nm exhibits a phenomenon of first increasing and then decreasing. When the TMB concentration reaches 0.225 mmol / L, the reaction system exhibits the maximum absorbance, and then, as the TMB concentration continues to increase, the absorbance of the reaction system gradually weakens. Based on this, it can be concluded that using 0.225 mmol / L TMB as the reaction system condition in the embodiments of this disclosure can obtain the best results.
[0094] Figure 11 This diagram illustrates the effect of different Ag₂O concentrations on the absorbance values of the reaction system in embodiments of this disclosure. Figure 11 As shown, in the embodiments of this disclosure, the Ag₂O concentration is between 50 μmol / L and 150 μmol / L. According to... Figure 11 It can be seen that as the Ag₂O concentration gradually increases, the absorbance value of the reaction system at 652 nm shows a trend of first increasing and then decreasing. The absorbance reaches its maximum value when the Ag₂O concentration is 125 μmol / L. Therefore, in this embodiment, the optimal Ag₂O concentration in the reaction system is 125 μmol / L.
[0095] Figure 12 This diagram illustrates the effect of different reaction times on the absorbance value of the reaction system in embodiments of this disclosure. Figure 12 As shown in the embodiments of this disclosure, the effects of reaction times of 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min on the absorbance values of the reaction system were selected. According to... Figure 12 It can be seen that as the reaction time increases, the absorbance value of the reaction system at 652 nm shows a trend of first increasing and then decreasing. When the system reacts for 50 minutes, the absorbance value reaches its maximum value. Based on this, it can be concluded that using a reaction time of 50 minutes as the reaction system condition in the embodiments of this disclosure can obtain the best results.
[0096] Figure 13 This is the ultraviolet absorption spectrum of the Ag2O-H2O2-TMB detection system in an embodiment of this disclosure. Figure 13 As shown, in this embodiment of the present disclosure, H2O2 standard solutions of different concentrations were prepared, and under optimal conditions, the H2O2 standard solutions were added to a solution containing Ag2O nanoparticles and TMB. The H2O2 was measured according to the method described in this embodiment of the present disclosure, and the absorbance value at 652 nm was measured using a T2600 UV spectrophotometer. Figure 13 It can be seen that as the H₂O₂ concentration increases from 0 μmol / L to 100 μmol / L, the absorbance of this reaction system gradually increases. It should be noted that... Figure 13In the study, the concentrations of Ag₂O were 125 μmol / L, TMB were 0.225 mmol / L, and the concentrations of AF:H₂O₂ were 0 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 50 μmol / L, and 100 μmol / L, respectively.
[0097] Figure 14 According to Figure 13 The linear fit plot of the detection system in this embodiment is drawn from the absorbance values in the ultraviolet spectrum. Figure 14 As shown, the linear equation of the standard curve obtained in this embodiment is A = 0.2212 + 0.00176C, the correlation coefficient R² = 0.9533, and the detection limit is 0.33 μmol / L. It should be noted that... Figure 14 The concentrations of Ag₂O were 125 μmol / L, TMB was 0.225 mmol / L, and H₂O₂ were 1 μmol / L, 5 μmol / L, 10 μmol / L, 50 μmol / L, and 100 μmol / L, respectively.
[0098] Figure 15 This is a schematic diagram illustrating the effect of interfering ions on the detection system in an embodiment of this disclosure. For example... Figure 15 As shown, in order to evaluate the selectivity of the Ag2O-H2O2-TMB detection system, this embodiment of the present disclosure selected five common metal ions (K... + Pb 2+ Cu 2+ Ni 2+ and Fe 2+ The effect of H2O2 detection on the detection was determined. Under optimal conditions, the concentration of H2O2 in this embodiment was selected as 10 μmol / L, and K... + Pb 2+ Cu 2+ Ni 2+ and Fe 2+ The concentrations of both were 100 μmol / L, and the concentrations of H2O2 and K were measured. + Pb 2+ Cu 2+ Ni 2+ and Fe 2+ The effect of a single substance on the absorbance value of the measurement system was investigated, and the standard deviation was found to be less than 10%. Furthermore, based on... Figure 15 It can be seen that ten times K + Pb 2+ Cu 2+ Ni 2+ and Fe 2+The method does not interfere with the detection of H2O2 in the embodiments of this disclosure. Therefore, it can be seen that the colorimetric method for detecting H2O2 provided in the embodiments of this disclosure has good selectivity for H2O2 detection.
[0099] This disclosure also includes an analysis of H2O2 in actual samples, the specific procedures of which are as follows:
[0100] Under optimal experimental conditions, this disclosure establishes a method for detecting H2O2 based on octahedral Ag2O nanozymes, and applies this method to the detection of H2O2 in milk. To reduce interference from other proteins in milk, the milk was diluted 10-fold with a buffer solution (pH 4.5). The results of detecting H2O2 in milk using the octahedral Ag2O nanozyme method described in this disclosure are shown in Table 1 below:
[0101] Table 1: Results of examining H2O2 in milk using the colorimetric method for detecting H2O2 provided in this embodiment of the present disclosure.
[0102]
[0103] According to the data in Table 1:
[0104] The colorimetric method for detecting H2O2 provided in this disclosure achieves a recovery rate of 88%–95% and a relative standard deviation of less than 5%. Therefore, this method can be used for detecting H2O2 in actual samples such as dairy products, demonstrating significant application value for food safety testing. It should be noted that the colorimetric method for detecting H2O2 in this disclosure can also be used to detect H2O2 in beverages and honey.
[0105] In summary, Ag₂O nanomaterials were prepared via a room-temperature ammonia method in this embodiment, and their morphology and structure were characterized by SEM and XRD, revealing an octahedral structure in the Ag₂O nanoparticles. Using octahedral Ag₂O nanozymes as catalysts, H₂O₂ was oxidized to TMB to generate oxTMB, changing the solution color from colorless to blue. Based on this, a simple, efficient, and accurate method for detecting H₂O₂ was successfully established. Furthermore, utilizing the mimicking oxidase activity of Ag₂O nanozymes, a novel colorimetric sensor for detecting H₂O₂ was constructed. This sensor was used to implement the colorimetric detection method for H₂O₂ in this embodiment. Under optimal reaction conditions, the recovery rate of H₂O₂ in physical samples was between 88% and 95%, with a relative standard deviation of less than 5%. This indicates that the colorimetric method for detecting H₂O₂ provided in this embodiment has good practical detection performance. This detection method has advantages such as simple operation, accurate detection, and visualized results, and has broad application prospects.
[0106] Those skilled in the art will understand that all or part of the steps of the above-described method embodiments can be implemented by hardware related to program instructions. The aforementioned program can be pre-installed in a computer-readable storage medium. When executed, the program performs the steps of the above-described method embodiments; and the aforementioned storage medium includes various media capable of storing program code, such as ROM, RAM, magnetic disks, or optical disks.
[0107] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the following claims.
[0108] It should be understood that this disclosure is not limited to the precise structures described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of this disclosure is limited only by the appended claims.
Claims
1. An octahedral A g2 The method for preparing O nanozymes is characterized by... The method includes the following steps: Step S1: Prepare nitric acid solution, ammonia solution and sodium hydroxide solution; Step S2: Slowly add the ammonia solution to the nitric acid solution to form a silver ammonia solution and stir for a preset time. While stirring, add the sodium hydroxide solution to the mixture dropwise. After a large amount of brown precipitate is formed, leave the precipitate in the solution for a preset time, collect and wash the precipitate to obtain octahedral Ag2O nanozyme powder. Step S3: Characterize the Ag2O nanozyme powder using X-ray diffraction; Step S4: Characterize the morphology and size of the Ag2O nanozyme powder using scanning electron microscopy.
2. The method according to claim 1, characterized in that, In step S1, The preparation of silver nitrate solution includes: weighing 0.8494 g of silver nitrate into a beaker and adding 45 mL of pure water and stirring until fully dissolved. Then, transfer the silver nitrate solution to a 50 mL volumetric flask, make up to volume with a dropper, and label the flask to obtain a silver nitrate solution with a concentration of 0.1 mol / L. The preparation of an ammonia solution involves: taking 376 μL of 25% ammonia solution, adding sufficient pure water to dilute it into a 50 mL volumetric flask, and obtaining an ammonia solution with a concentration of 0.1 mol / L; The preparation of sodium hydroxide solution includes: weighing 2.000 g of sodium hydroxide into a beaker and adding 20 mL of pure water, stirring continuously until the sodium hydroxide is completely dissolved. Then, transfer the sodium hydroxide solution to a 25 mL volumetric flask, dilute to volume with a dropper, and label the flask to obtain a sodium hydroxide solution with a concentration of 2.0 mol / L.
3. The method according to claim 1, characterized in that, In step S2, 50 mL of 0.1 mol / L AgNO3 solution was placed in a beaker. Under constant temperature and magnetic stirring at 600 r / min, 25 mL of 0.1 mol / L ammonia water was slowly added dropwise to form a silver ammonia solution. After stirring for 10 min, 2.5 mL of 2 mol / L NaOH solution was added dropwise to the mixture while stirring. After a large amount of brown precipitate was formed, the precipitate was left in the solution for 12 h. The precipitate was then centrifuged at 8000 rpm for 25 min. The precipitate was collected and washed with ethanol and pure water until the pH of the final aqueous solution was neutral, yielding octahedral Ag2O nanozyme powder.
4. The method according to claim 1, characterized in that, In step S3, The octahedral Ag2O nanozyme powder was dispersed in a square frosted area of a glass slide and spread evenly. After being flattened with a coverslip, it was characterized by X-ray diffraction.
5. The method according to claim 4, characterized in that, In step S4, The conductive adhesive was fixed on the sample stage. The octahedral Ag2O nanozyme powder was ground into powder and then dotted onto the conductive adhesive. After being blown and spread evenly, it was placed in a scanning electron microscope for inspection and photographs were taken.
6. A colorimetric method for detecting H2O2, characterized in that, The method includes the following steps: Step S11: Take several centrifuge tubes and add a measured amount of A to each centrifuge tube. g2 O solution and H2O2 of different concentrations are mixed evenly; wherein, the A g2 Solution O is the octahedral A according to any one of claims 1-5 g2 It was prepared using the method for preparing O nanozymes; Step S12: After adding NaAc-Hac buffer and TMB solution to each centrifuge tube processed in step S11, an Ag2O-TMB-H2O2 reaction system is formed. After heating the Ag2O-TMB-H2O2 reaction system in each centrifuge tube in a water bath for a preset time, the color change of the solution in each centrifuge tube is observed. Step S13: Measure the absorbance of the Ag2O-TMB-H2O2 reaction system at a preset wavelength using an ultraviolet spectrophotometer and record the data.
7. The method according to claim 6, characterized in that, Before step S12, the method further includes: preparing NaAc-Hac buffer and TMB solution; wherein, The preparation of NaAc-Hac buffer solution includes: weighing 4.1017 g of anhydrous sodium acetate into a container, adding 2.86 mL of glacial acetic acid, and then adding sufficient pure water to prepare 500 mL of 0.1 mol / L buffer solution; The preparation of the TMB solution involves: weighing 0.2500 g of 3,3′,5,5′-tetramethylbenzidine into a container, then adding 1.5 mL of anhydrous ethanol, heating and stirring constantly until completely dissolved, and then making up to 25 mL in a volumetric flask to obtain a 41.6 mmol / L TMB solution.
8. The method according to claim 6, characterized in that, The method includes: In step S11: Take several 5 mL centrifuge tubes and add 50 μL of A to each centrifuge tube. g2 O solution and 20 μL of H2O2 of different concentrations are mixed evenly; In step S12: 3810 μL of 0.1 mol / L NaAc-Hac buffer solution with pH 4.5 and 120 μL of TMB solution were added to each centrifuge tube after the treatment in step S11 to form an Ag2O-TMB-H2O2 reaction system. After heating the Ag2O-TMB-H2O2 reaction system in each centrifuge tube in a water bath at 25 ℃ for 50 min, the color change of the solution in each centrifuge tube was observed with the naked eye. In step S13, the absorbance of the Ag2O-TMB-H2O2 reaction system at a wavelength of 652 nm is measured using a T2600 UV spectrophotometer and the data is recorded.
9. The method according to claim 6, characterized in that, The method further includes the following steps: Step S14: Measure 5 mL of the test solution into a 50 mL beaker using a graduated cylinder, add 45 mL of 0.1 mol / L NaAc-HAc buffer solution and stir to mix well. After standing for 15 min, centrifuge at 8000 r / min for 20 min and take the supernatant as the solvent to prepare the spiked sample of H2O2. Step S15: Add the spiked sample to a NaAc-HAc buffer solution containing Ag2O nanozyme and TMB solution to form a reaction solution; wherein, in the reaction solution, the concentration of Ag2O nanozyme is 125 μmol / L, the concentration of TMB is 0.225 mmol / L, and the concentrations of H2O2 in the spiked sample are 5 μmol / L, 10 μmol / L, and 50 μmol / L, respectively; In step S16, after heating the solution treated in step S15 in a water bath at 25 ℃ for 50 min, the absorption spectrum of the reaction solution at 652 nm was measured using a T2600 UV spectrophotometer and the data was recorded. The recovery rate was then calculated.