Preparation method of ultrathin two-dimensional oxygen vacancy-rich cobalt oxide and application of ultrathin two-dimensional oxygen vacancy-rich cobalt oxide in food total antioxidant capacity detection

Abstract

The invention relates to a preparation method of ultrathin two-dimensional cobalt oxide rich in oxygen vacancies and an application of the ultrathin two-dimensional cobalt oxide rich in oxygen vacancies in food total antioxidant capacity detection, and belongs to the fields of low-dimensional nano materials, biological mimic enzyme catalysis and analytical chemistry. According to the preparation method, cobalt acetylacetonate and hexadecyl trimethyl ammonium bromide are adopted as raw materials, and the ultrathin two-dimensional cobalt oxide nano-enzyme rich in oxygen vacancies is prepared through a hydrothermal reaction and high-temperature pyrolysis. The obtained ultrathin two-dimensional cobalt oxide nano-enzyme rich in oxygen vacancies has excellent peroxidase-like catalytic activity and selectivity. The oxygen-vacancy-rich metal oxide has the advantages of high stability, easy mass production and low preparation cost, and is expected to be widely used in the fields of biosensing, agriculture, food engineering, catalysis and the like.

Description

Technical Field

[0001] This invention relates to the fields of low-dimensional nanomaterials, biomimetic enzyme catalysis, and analytical chemistry, and particularly to a method for preparing ultrathin two-dimensional cobalt oxide rich in oxygen vacancies and its application in the detection of total antioxidant capacity in food. Background Technology

[0002] Ascorbic acid (AA), also known as vitamin C, is a natural, water-soluble organic compound widely distributed in vegetables and fruits. As an essential micronutrient, ascorbic acid plays a crucial role in various physiological and biochemical processes, including promoting the absorption of non-heme iron, regulating immune function, promoting collagen biosynthesis, and scavenging reactive oxygen species (ROS) and free radicals, thus playing a vital role in maintaining the body's redox balance. Furthermore, ascorbic acid acts as a cofactor in many enzymatic reactions, playing an irreplaceable role in the body's metabolic processes. Since humans cannot synthesize ascorbic acid endogenously, they must rely on exogenous intake, such as from fresh fruits and vegetables and nutritional supplements. Insufficient ascorbic acid intake can lead to scurvy, cardiovascular and cerebrovascular diseases, and other pathological conditions related to oxidative stress. Therefore, establishing a sensitive, rapid, and efficient method for ascorbic acid detection is of significant practical and clinical importance for ensuring food safety, assessing nutritional components, and diagnosing diseases. Currently, there are various methods for detecting ascorbic acid, covering multiple technical pathways such as electrochemical analysis, high-performance liquid chromatography, fluorescence analysis, colorimetric analysis, chemiluminescence, and electrochemiluminescence. Among them, colorimetric analysis has attracted much attention due to its simplicity, low cost, on-site analysis, and rapid detection. In particular, colorimetric sensor technology based on nanozymes is considered an ideal alternative to traditional enzymes due to its convenient construction, high stability, and controllable cost.

[0003] Nanozymes are a class of nanoscale materials with catalytic functions similar to natural enzymes. In recent years, they have gradually become a frontier in enzyme-mimicking catalysis research due to their advantages such as low cost, high stability, tunable catalytic activity, and ease of large-scale production. Two-dimensional ultrathin metal oxide nanozymes, with their unique size effect, significantly increased specific surface area, and electronic structure different from bulk materials, have shown broad application prospects in multiple fields such as biosensing, food safety, and disease diagnosis and treatment. Their high proportion of exposed surface atoms makes it possible to construct surface defects, which in turn facilitates the regulation and creation of catalytic active sites. These structural features not only help to form new reactive centers in enzyme-like catalysis but also effectively regulate electron transport behavior, thus providing a new strategy for the controllable regulation of nanozyme catalytic performance. Therefore, how to use defect engineering methods, especially by introducing surface oxygen vacancies to precisely control the surface electronic state and adsorption properties of materials, thereby further optimizing their enzyme-mimicking catalytic performance, has become a key scientific and technological challenge that urgently needs to be overcome in the current research on nanozyme design and application. Summary of the Invention

[0004] To address the aforementioned problems, the main objective of this invention is to provide a method for preparing ultrathin two-dimensional cobalt oxide rich in oxygen vacancies and its application in ascorbic acid detection. The ultrathin two-dimensional cobalt oxide rich in oxygen vacancies exhibits excellent peroxidase-like catalytic activity, and a colorimetric sensor constructed using this cobalt oxide as a catalyst can achieve highly sensitive and specific detection of ascorbic acid concentration in food.

[0005] To achieve the above and other related objectives, the present invention provides the following technical solution: a method for preparing ultrathin two-dimensional cobalt oxide rich in oxygen vacancies, comprising: preparing ultrathin cobalt-based nanosheets by hydrothermal method, and then obtaining ultrathin two-dimensional cobalt oxide nanosheets rich in oxygen vacancies after high-temperature pyrolysis.

[0006] The preferred technical solution includes the following steps: Step 1: Add hexadecyltrimethylammonium bromide to a mixed solution of ethanol and deionized water, stir, and then add cobalt acetylacetone to obtain a precursor solution; transfer the precursor solution to a high-pressure reactor, and generate ultrathin cobalt-based nanosheets after hydrothermal reaction. Step 2: Using ultrathin cobalt-based nanosheets as templates, ultrathin two-dimensional cobalt oxide nanosheets rich in oxygen vacancies are obtained by pyrolysis under an inert gas atmosphere.

[0007] The preferred technical solution is that the ratio of hexadecyltrimethylammonium bromide to cobalt acetylacetonate is 1~4g: 20~120mg.

[0008] The preferred technical solution is as follows: the hydrothermal reaction process parameters are: temperature 80~300℃, reaction time 6~72 hours.

[0009] The preferred technical solution is as follows: in step 2, the inert gas is nitrogen or argon; the pyrolysis temperature is 100~500℃, and the pyrolysis holding time is 10~180min.

[0010] To achieve the above and other related objectives, the present invention provides the following technical solution: The application of ultrathin two-dimensional cobalt oxide rich in oxygen vacancies in the detection of total antioxidant capacity in food, characterized by comprising the following steps: S1. The ultrathin two-dimensional cobalt oxide rich in oxygen vacancies, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 solution were added to NaAc-HAc buffer solution to obtain a mixed solution. The mixed solution was incubated and the absorbance of the mixed solution at 652 nm was measured. S2. Mix the ascorbic acid solutions of different concentrations with the mixed solution obtained in S1, then incubate them, measure the absorbance at 652 nm, and draw a linear equation about the ascorbic acid concentration and absorbance value to achieve quantitative detection. S3. Mix the sample solution to be tested with the mixed solution obtained in S1, then incubate, measure the absorbance at 652 nm, and calculate the concentration of ascorbic acid in the sample to be tested.

[0011] The preferred technical solution is as follows: the detection range of ascorbic acid is 0-1400 μM, and the detection limit is 0.003 μM; the pH value of the NaAc-HAc buffer is 3.5-4.5, the concentration of cobalt oxide nanozyme is 0.1-4 mg / ml, the concentration of ascorbic acid solution is 0-20 mM, the concentration of TMB solution is 1-20 mM, and the concentration of H2O2 solution is 1-1.2 mM.

[0012] The preferred technical solution is: the incubation temperature is 20-50℃, and the incubation time is 1-10 min. Beneficial effects

[0013] By employing the above-described technical solution, the advantages of this invention compared to the prior art are: 1. This invention uses hexadecyltrimethylammonium bromide and cobalt acetylacetonate as raw materials to prepare ultrathin cobalt-based nanosheets via hydrothermal reaction, followed by high-temperature pyrolysis to obtain ultrathin two-dimensional cobalt oxide nanoenzymes rich in oxygen vacancies. This invention utilizes defect engineering techniques to precisely control the oxygen vacancy concentration on the surface of the ultrathin two-dimensional cobalt oxide material, optimizing the surface electronic states and adsorption properties of the cobalt oxide material, accelerating the enzyme-mimicking catalytic reaction rate, and improving the performance of enzyme-mimicking catalysis.

[0014] 2. The ultrathin two-dimensional cobalt oxide nanozyme rich in oxygen vacancies provided by this invention catalyzes the oxidation of TMB in the presence of hydrogen peroxide, producing a distinct color and exhibiting excellent peroxidase-like activity. When ascorbic acid is introduced into the system, the oxidized TMB is reduced, and the solution color gradually fades from blue. The absorbance change during this process shows a good linear correlation with the ascorbic acid concentration, and the detection limit of the method is as low as 0.003 μM, demonstrating extremely high detection sensitivity. This invention establishes a highly sensitive, rapid-response, and simple-to-operate colorimetric method that can be used for the accurate analysis of ascorbic acid content in food. This method shows good application potential and broad prospects for promotion in the fields of food component detection and quality and safety control. Attached Figure Description

[0015] Figure 1 CoO in Embodiment 1 of the present invention 2x Transmission electron microscope image.

[0016] Figure 2CoO in Embodiment 1 of the present invention 2x X-ray diffraction pattern.

[0017] Figure 3 CoO in Embodiment 1 of the present invention 2x An atomic force microscope image.

[0018] Figure 4 CoO in Embodiment 1 of the present invention 2x O 1s X-ray photoelectron spectrum.

[0019] Figure 5 This refers to the CoO from Embodiment 3, Comparative Example 1, and Comparative Example 2 of the present invention. 2x A diagram illustrating the effect of mimicking peroxidase activity.

[0020] Figure 6 This is the CoO-based embodiment of the present invention, which is described in Example 4. 2x Quantitative detection graph of ascorbic acid by colorimetric sensing in the catalytic system.

[0021] Figure 7 This is the CoO from Embodiment 5 of the present invention. 2x Comparison of the selectivity of catalysts in the detection of ascorbic acid.

[0022] Figure 8 This is the CoO-based embodiment of the present invention, which is described in Example 6. 2x A graph showing the detection of the total antioxidant capacity of catalysts in food. Detailed Implementation

[0023] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0024] Please see Figure 1-8 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and are not intended to limit the implementation of the invention. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size, without affecting the effectiveness or purpose of the invention, should fall within the scope of the disclosed technical content. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional consumables and biochemical reagent stores.

[0025] Example 1: Ultrathin two-dimensional cobalt oxide nanosheets rich in oxygen vacancies (CoO2) 2x Preparation of ) 1) Preparation of ultrathin cobalt-based nanosheets: 2.2 g of hexadecyltrimethylammonium bromide was dissolved in a mixed solution of 60 ml ethanol and 20 ml deionized water. After stirring until the reaction solution was clear, 60 mg of cobalt acetylacetonate powder was added and ultrasonically dispersed evenly. The above precursor solution was transferred to a high-pressure reactor and reacted at 180 °C for 48 h. The reaction solution was then removed and washed alternately by centrifugation with a mixed solution of deionized water and ethanol to obtain ultrathin cobalt-based nanosheets.

[0026] 2) Take 30 mg of the ultrathin cobalt-based nanosheet powder obtained in step 1), place it in a tube furnace, and introduce 99.999% argon gas into the furnace tube at a flow rate of 50 mL / min. Then, heat the tube furnace to 350 °C at a heating rate of 5 °C / min, hold it at that temperature for 1 h, and then cool it to room temperature to obtain the ultrathin two-dimensional cobalt oxide material rich in oxygen vacancies.

[0027] See appendix for material morphology. Figure 1 This indicates that the ultrathin two-dimensional cobalt oxide rich in oxygen vacancies possesses a layered structure. (See attached image.) Figure 2 As shown, the X-ray diffraction patterns correspond to the (200) crystal plane at 42.8°, the (220) crystal plane at 62.2°, and the (220) crystal plane at 76.8°, respectively. However, no obvious diffraction peaks were observed for the (111) and (311) crystal planes, which is related to the missing crystal planes caused by the loss of some dimensions in the two-dimensional material. (See attached image) Figure 3 As shown, the thickness of the ultrathin two-dimensional cobalt oxide nanosheets rich in oxygen vacancies, as observed by atomic force microscopy, is 0.73 nm, approximately 5 atomic layers thick, demonstrating its ultrathin structural characteristics. (See attached image.) Figure 4 As shown, X-ray photoelectron spectroscopy indicates that the oxygen vacancy rate on the surface of the ultrathin two-dimensional cobalt oxide sample rich in oxygen vacancies is 51.3%, indicating the presence of a large number of oxygen vacancies on the surface of the ultrathin cobalt oxide atomic layer.

[0028] Example 2: Ultrathin two-dimensional cobalt oxide nanosheets with oxygen vacancies (CoO2) x Preparation of ) 1) Preparation of ultrathin cobalt-based nanosheets: 2.2 g of hexadecyltrimethylammonium bromide was dissolved in a mixed solution of 60 ml ethanol and 20 ml deionized water. After stirring until the reaction solution was clear, 60 mg of cobalt acetylacetonate powder was added and ultrasonically dispersed evenly. The above precursor solution was transferred to a high-pressure reactor and reacted at 180 °C for 48 h. The reaction solution was then removed and washed alternately by centrifugation with a mixed solution of deionized water and ethanol to obtain ultrathin cobalt-based nanosheets.

[0029] 2) Take 30 mg of the ultrathin cobalt-based nanosheet powder obtained in step 1), place it in a tube furnace, and introduce 99.999% argon gas into the furnace tube at a flow rate of 50 mL / min. Then, heat the tube furnace to 450℃ at a heating rate of 5℃ / min, hold it at that temperature for 1 h, and then cool it to room temperature to obtain the ultrathin two-dimensional cobalt oxide material containing oxygen vacancies.

[0030] Example 3: Verification of the peroxidase-like activity of ultrathin two-dimensional cobalt oxide nanozymes rich in oxygen vacancies The catalytic reaction system contains H2O2 (20 ml, 10 mM) and CoO obtained in Example 1. 2x The reaction mixture consisted of 20 µl (200 mg / mL), organic chromogenic reagent TMB (20 mL, 6 mM), and pH 4 buffer (140 mL, 10 mM). The reaction was carried out at room temperature (25 °C) for 10 minutes.

[0031] Comparative Example 1 The catalytic reaction system and operation are similar to those in Example 3, except that the catalytic reaction system does not contain CoO. 2x Nanocomposite.

[0032] Comparative Example 2 The catalytic reaction system and operation are similar to those in Example 3, except that the catalytic reaction system does not contain H2O2.

[0033] Example 3 (CoO) 2x + H2O2+TMB), Comparative Example 1 (H2O2+TMB) and Comparative Example 2 (CoO2+TMB) 2x The absorbance of the solution obtained after the reaction with TMB was measured in the range of 500-800 nm using an ELISA reader under the same conditions.

[0034] Figure 5 This refers to the CoO from Embodiment 3, Comparative Example 1, and Comparative Example 2 of the present invention. 2x A diagram illustrating the effect of mimicking peroxidase activity.

[0035] like Figure 5 As shown, the solution sample obtained in Example 3 exhibits a significant absorption peak of the colorimetric reagent oxidation product near 652 nm, indicating that CoO 2x It exhibits significant peroxidase-like activity at pH 4; Comparative Example 1 shows no absorption peak near 652 nm, indicating that without CoO2... 2x The nanocomposite as a catalyst will not produce a color reaction; Comparative Example 2 shows no obvious absorption peak near 652 nm, indicating that the absorption peak of the solution sample in Example 5 is not CoO. 2x Its own response peak.

[0036] Example 4: Ultrathin two-dimensional cobalt oxide nanozyme biosensor rich in oxygen vacancies for ascorbic acid detection The catalytic reaction system contained different concentrations of ascorbic acid (20 ml, 0 mM, 0.1 mM, 0.3 mM, 0.7 mM, 1 mM, 2 mM, 3 mM, 4 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM) and CoO. 2x The ascorbic acid standard curve was prepared using 20 µl (200 mg / mL), H2O2 (20 µl, 20 mM), TMB (20 ml, 6 mM), and pH 4 buffer (140 ml, 10 mM). The reaction was carried out at 25 °C for 30 minutes. The absorbance at 652 nm was measured using a microplate reader, and a standard working curve was plotted.

[0037] Figure 6 This is Embodiment 4 of the present invention based on CoO 2x Quantitative detection chromatogram of ascorbic acid in the catalytic system using colorimetric sensing, as shown in... Figure 6 As shown, the linear range is 0-1400 mM, Abs. = 1.0648 - 0.7579C (R 2 = 0.9971).

[0038] Example 5: Ultrathin two-dimensional cobalt oxide nanozyme biosensor rich in oxygen vacancies with selective... Selectivity experiment: 20 ml of 0.5 mM ascorbic acid and 5 mM phenylalanine, cysteine, histidine, glutamic acid, and Zn were taken respectively. 2+ Mg 2+ K + Na + Fructose, sucrose, and maltose are added to the catalytic reaction system, wherein the reaction system contains CoO 2x The reaction mixture consisted of 20 µl (200 mg / mL), chromogenic reagent TMB (20 ml, 6 mM), and pH 4 buffer (120 ml, 10 mM). The reaction was carried out at 25°C for 30 minutes, and the absorbance at 652 nm was measured using a microplate reader.

[0039] Figure 7 This is the CoO from Embodiment 5 of the present invention. 2x A comparison chart of the selectivity of catalysts in the detection of ascorbic acid. (e.g.) Figure 7 As shown, from left to right, they are phenylalanine, cysteine, histidine, glutamic acid, and Zn. 2+ Mg 2+ K + Na +Fructose, sucrose, and maltose. Even when the concentration of ascorbic acid in the control group was 10 times higher than that of glucose, the absorbance of ascorbic acid was still much lower than that of the control group, indicating that it is based on CoO2. 2x The biosensor constructed from nanozymes exhibits high selectivity for the detection of ascorbic acid.

[0040] Example 6: Application of ultrathin two-dimensional cobalt oxide nanozymes rich in oxygen vacancies in the detection of total antioxidant capacity in food. Using ascorbic acid as a typical model compound, the total antioxidant capacity of various commercially available beverages (including green tea, iced black tea, and Nongfu Spring beverages) was quantitatively evaluated. The total antioxidant capacity was measured under the same experimental conditions as the standard ascorbic acid test, except that actual beverage samples were used instead of the added ascorbic acid solution. It is particularly important to note that to ensure the accuracy of the test results and consistency with the linear response range of the method, the concentration of the actual samples must be appropriately diluted and adjusted to avoid their antioxidant capacity exceeding the effective detection range of the standard curve.

[0041] As attached Figure 8 As shown, Farmer Orchard had the highest antioxidant content among the three commercial beverages.

[0042] In summary, this invention provides an ultrathin, two-dimensional, oxygen-vacancy-rich cobalt oxide nanozyme with peroxidase-like catalytic activity, as well as its preparation and application. The ascorbic acid colorimetric sensing platform constructed based on this nanozyme exhibits a broad linear detection range, extremely low detection limit, excellent catalytic performance, and good selective recognition ability. These research results fully demonstrate that this type of ultrathin, two-dimensional, oxygen-vacancy-rich cobalt oxide biomimetic catalytic material has significant application potential and promotional value in the field of high-sensitivity ascorbic acid detection.

[0043] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A method for preparing an ultrathin two-dimensional cobalt oxide rich in oxygen vacancies, characterized in that: The application relates to a method for preparing a two-dimensional cobalt oxide nanosheet with rich oxygen vacancies. The method comprises the following steps:

2. The method of claim 1, wherein the method is characterized by: Step 1: adding cetyltrimethylammonium bromide into a mixed solution composed of ethanol and deionized water, stirring, and then adding cobalt acetylacetonate to obtain a precursor solution; transferring the precursor solution into a high-pressure reaction kettle, and generating the ultra-thin cobalt-based nanosheet after hydrothermal reaction; Step 2: taking the ultra-thin cobalt-based nanosheet as a template, and obtaining the ultra-thin two-dimensional cobalt oxide nanosheet with rich oxygen vacancies through pyrolysis in an inert gas atmosphere. The ratio of cetyltrimethylammonium bromide to cobalt acetylacetonate is 1-4 g: 20-120 mg.

3. The method of claim 2, wherein the method is characterized by: The hydrothermal reaction process parameters are as follows: the temperature is 80-300 DEG C, and the reaction time is 6-72 hours.

4. The method of claim 2, wherein the method is characterized by: In step 2, the inert gas is nitrogen or argon; the pyrolysis temperature is 100-500 DEG C, and the pyrolysis holding time is 10-180 min.

5. The method of claim 2, wherein the method is characterized by: The method comprises the following steps:

6. The use of the ultrathin two-dimensional cobalt oxide rich in oxygen vacancies according to any one of claims 1-5 in the detection of the total antioxidant capacity of food, characterized by: S1, adding the ultra-thin two-dimensional cobalt oxide nanosheet with rich oxygen vacancies, a 3,3',5,5'-tetramethylbenzidine solution and an H2O2 solution into a NaAc-HAc buffer solution respectively to obtain a mixed solution, incubating the obtained mixed solution, and measuring the absorbance of the mixed solution at 652 nm; S2, mixing ascorbic acid solutions with different concentrations with the mixed solution obtained in S1, then incubating, measuring the absorbance at 652 nm, and drawing a linear equation about the ascorbic acid concentration and the absorbance value to realize quantitative detection; S3, mixing a sample solution to be detected with the mixed solution obtained in S1, then incubating, measuring the absorbance at 652 nm, and calculating the ascorbic acid concentration in the sample solution to be detected. The ascorbic acid detection range is 0-1400 muM, and the detection limit is 0.003 muM; the pH value of the NaAc-HAc buffer solution is 3.5-4.5, the concentration of the cobalt oxide nanosheet is 0.1-4 mg / ml, the concentration of the ascorbic acid solution is 0-20 mM, the concentration of the TMB solution is 1-20 mM, and the concentration of the H2O2 solution is 1-1.2 mM.

7. Use of the ultrathin two-dimensional cobalt oxide rich in oxygen vacancies according to claim 6 in the detection of the total antioxidant capacity of foods, characterized by: The incubation temperature is 20-50 DEG C, and the incubation time is 1-10 min.

8. Use of the ultrathin two-dimensional oxygen-vacancy-rich cobalt oxide according to claim 6 in the detection of total antioxidant capacity of food, characterized in that: ​

Images