A hollow Fe3O4@MnO2 flower-shaped nanozyme, its preparation method, and its application in vitamin C content detection.

Abstract

The application provides a hollow Fe3O4@MnO2 flower-shaped nanoscale enzyme, a preparation method and application thereof in vitamin C content detection, the nanoscale enzyme comprises a hollow core and a coated outer layer, the hollow core is a hollow Fe3O4 sphere, and the coated outer layer is an MnO2 layer; the overall structure is hollow flower-shaped, has high catalytic activity and stability, and can be used for detecting the content of vitamin C; the hollow Fe3O4@MnO2 flower-shaped nanoscale enzyme can effectively widen the detection range of vitamin C, improve detection accuracy and reduce the detection limit, and is helpful for quickly, simply and accurately determining the vitamin C content of various to-be-detected substances.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a hollow Fe3O4@MnO2 flower-shaped nanozyme, its preparation method, and its application in the detection of vitamin C content. Background Technology

[0002] Vitamin C (VC) is a common vitamin beneficial to the human body. Studies have shown that vitamin C has antioxidant properties, promotes collagen synthesis, delays cell aging and apoptosis, and can increase the body's immunity and resistance. However, both vitamin C deficiency and excess are detrimental to health. Therefore, detecting the vitamin C content in analytes containing vitamin C is of great significance.

[0003] Currently, the main methods for detecting vitamin C content are high-performance liquid chromatography (HPLC), electrochemical methods, and ultraviolet-visible spectrophotometry (UV-Vis). HPLC requires complex sample pretreatment, relies on large benchtop instruments, and is time-consuming. Electrochemical methods require precise analytical instruments and are easily affected by external interference. UV-Vis spectrophotometry has many interfering factors and poor selectivity.

[0004] Natural enzymes possess good specificity, but they are expensive and easily deactivated. Nanozymes are nanomaterials with catalytic activity similar to natural enzymes. Nanozymes with excellent oxidase-like activity can be used in combination with specific substrates, such as 3,3',5,5'-tetramethylbenzidine (TMB), to catalyze the formation of a blue oxidation product (TMB) from colorless TMB. OX Vitamin C, with its strong reducing properties, can reduce oxidation products, causing them to fade from blue to colorless. The resulting absorbance and color change can be used to measure the vitamin C content in the analyte. However, different nanozymes have varying catalytic activities and stability, resulting in differences in detection range, detection limit, and accuracy when applied to vitamin C detection, making it difficult to meet the detection requirements of wide concentration range and high sensitivity in different scenarios.

[0005] Therefore, there is an urgent need to design a nanozyme with high catalytic activity and high stability, as well as a vitamin C detection method based on the nanozyme, to broaden the detection range of vitamin C, improve detection accuracy, and reduce the detection limit under low cost, convenience and speed conditions. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a hollow Fe3O4@MnO2 flower-shaped nanozyme, its preparation method, and its application in vitamin C content detection. The hollow Fe3O4@MnO2 flower-shaped nanozyme comprises a hollow core and a coating outer layer. The hollow core is a hollow Fe3O4 sphere, and the coating outer layer is a MnO2 layer. This nanozyme has a hollow flower-shaped structure and exhibits excellent oxidase-like catalytic activity and stability. Using it for vitamin C content detection can broaden the detection range of vitamin C content, improve detection accuracy, and lower the detection limit, thus facilitating the rapid, convenient, and accurate determination of vitamin C content in various analytes.

[0007] In existing technologies, different nanozymes exhibit varying catalytic activities and stability, resulting in differences in detection range, detection limit, and accuracy when applied to vitamin C detection. Therefore, this invention designs a nanozyme with high catalytic activity and high stability, achieving a broadened detection range, improved detection accuracy, and a lower detection limit for vitamin C under low-cost, convenient, and rapid conditions.

[0008] On one hand, the present invention provides a Fe3O4@MnO2 nanozyme, wherein the Fe3O4@MnO2 nanozyme comprises a hollow core and a coating outer layer, wherein the hollow core is a hollow Fe3O4 sphere; and the coating outer layer is a MnO2 layer, wherein the coating outer layer coats the hollow core to form a flower-like structure.

[0009] This application presents a hollow Fe3O4@MnO2 flower-shaped nanozyme designed through core-shell structure optimization, and its application in the detection of vitamin C content. The inventors compared this nanozyme with other nanozymes of the same composition, and the results showed that the hollow Fe3O4@MnO2 flower-shaped nanozyme exhibits a wider detection range, higher accuracy, and a lower detection limit when detecting vitamin C content, greatly meeting the needs for detecting different concentrations of vitamin C in various scenarios. Furthermore, its detection results showed no statistically significant difference from those obtained by liquid chromatography, but the detection time and difficulty were significantly reduced compared to liquid chromatography.

[0010] In the hollow Fe3O4@MnO2 flower-shaped nanoenzyme, the Fe3O4 spheres, acting as the hollow core, utilize their superparamagnetism to enable the entire composite material to be rapidly separated and recovered using an external magnetic field, solving the problem of difficult recovery and easy loss of pure MnO2 nanomaterials. The hollow structure provides loading sites and a larger specific surface area for the growth of the MnO2 shell, increasing the contact probability between the catalytic active sites and the reaction substrate, and synergistically enhancing the enzyme-like catalytic performance of the overall material.

[0011] The MnO2 layer itself possesses excellent oxidase-like catalytic activity and is the main contributor to the enzyme-like function of Fe3O4@MnO2 nanozymes. Simultaneously, as a shell encapsulating Fe3O4, it further enhances the dispersibility and stability of the entire core-shell structure in water treatment, detection, and other application environments, resulting in a longer lifespan. The hollow flower-shaped nanozyme formed by the combination of these two elements significantly broadens the detection range and improves the accuracy of vitamin C detection methods, while also offering convenient and rapid operation.

[0012] Furthermore, this invention provides a method for preparing hollow Fe3O4@MnO2 flower-shaped nanozymes, the preparation method comprising the following steps: (1) Preparation of hollow Fe3O4 spheres: FeCl3·6H2O and NH4Ac were dissolved, heated, washed and dried; (2) Preparation of hollow Fe3O4@MnO2 flower-shaped nanoenzymes: The synthesized hollow Fe3O4 spheres were prepared into a suspension, KMnO4 was added to the suspension, hydrochloric acid was added dropwise, the mixture was heated, washed and dried.

[0013] Furthermore, in the preparation method of the hollow Fe3O4@MnO2 flower-shaped nanozyme, the heating temperature for preparing the hollow Fe3O4 spheres is not lower than 200 ℃; and the heating time is not lower than 16 h.

[0014] Preferably, the heating temperature is 200 °C and the heating time is 16 h.

[0015] In the preparation of hollow Fe3O4 spheres, heating temperature and heating time are the core parameters. Too low a temperature or too short a heating time will prevent the formation of a hollow structure in the Fe3O4 spheres, while too high a temperature or too long a heating time may lead to a decrease in yield. Therefore, it is necessary to adjust the matching relationship between the two according to the target product. Through screening the heating temperature and heating time, the inventors found that the preferred heating temperature is 200 ℃ and the preferred heating time is 16 h, which yields the most stable hollow Fe3O4 spheres in terms of morphology and structure.

[0016] In another aspect, the present invention provides a method for detecting vitamin C using Fe3O4@MnO2 nanozymes, the method comprising the following steps: Fe3O4@MnO2 nanozyme, TMB solution, and buffer solution were uniformly mixed and reacted at room temperature. The analyte containing vitamin C was added, and the vitamin C content was calculated by spectrophotometry or colorimetry.

[0017] The nanozyme possesses oxidase-like activity, catalyzing the oxidation of the substrate 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen (O2) in an acidic system to generate a blue oxidation product (TMB). OXVitamin C, which has reducing properties, can reduce TMB. OX The color is reduced to a colorless state, allowing the vitamin C content to be calculated using spectrophotometry or colorimetry.

[0018] Furthermore, in the method for detecting vitamin C using Fe3O4@MnO2 nanozymes, the buffer solution is a NaAc-HAc buffer solution with a pH range of 3.5 to 4.5, and the TMB solution concentration range is 6 to 20 mM.

[0019] Preferably, the buffer solution has a pH of 4 and the TMB solution has a concentration of 6 mM.

[0020] In the method for detecting vitamin C using Fe3O4@MnO2 nanozymes, the pH value of the reaction system is one of the core parameters. It must be kept stable and within a suitable range, as the activity of oxidase-like enzymes is pH-dependent, and pH changes affect TMB. OX The stability of the buffer solution affects the detection results of vitamin C. After screening experiments, it was found that the oxidase activity was optimal when the pH of the buffer solution was 4, which is suitable for detection.

[0021] In the method for detecting vitamin C using Fe3O4@MnO2 flower-shaped nanozymes, the concentration of TMB has a significant impact on the detection performance. As a reaction substrate, if the initial TMB concentration is too low, even with sufficient catalytic reaction time, it will lead to TMB degradation. OX Limited TMB production narrows the linear detection range, and high-concentration vitamin C detection is prone to premature saturation, making accurate quantification impossible. Excessive TMB concentration increases background interference, enhances non-specific light absorption, and reduces sensitivity for low-concentration vitamin C detection; therefore, TMB concentration must be controlled within an appropriate range. This invention measures the absorbance at 652 nm of the system when TMB solution concentrations range from 0.1 to 20 mM. A TMB solution concentration of 6 mM was selected, at which point the absorbance at 652 nm tends to stabilize. Under these conditions, an optimal balance between detection accuracy and detection range can be achieved.

[0022] Preferably, in the above method for detecting vitamin C using Fe3O4@MnO2 nanozymes, the Fe3O4@MnO2 nanozyme has a hollow flower-like structure.

[0023] The nanozyme is a hollow Fe3O4@MnO2 flower-shaped nanozyme. Using this nanozyme for vitamin C detection offers advantages such as a wide detection range, high accuracy, and low detection limit. This application measured the absorption spectra of each system in the 500-700 nm range by mixing hollow Fe3O4@MnO2 flower-shaped nanozyme, hollow Fe3O4 spheres, and MnO2 nanosheets with TMB and NaAc-HAc buffer solutions. The results showed that the absorbance of the hollow Fe3O4@MnO2 flower-shaped nanozyme at 652 nm was significantly higher than that of the hollow Fe3O4 spheres and MnO2 nanosheets, indicating that the catalytic efficiency of the hollow Fe3O4@MnO2 flower-shaped nanozyme is significantly superior to the other two materials. Furthermore, due to the oxidation product TMB... OX The absorption peak is most prominent at 652 nm, so 652 nm was chosen as the measurement wavelength. This wavelength offers the highest sensitivity and is less sensitive to wavelength changes in the absorbance region at the top of the absorption peak. Consequently, the absorbance measurement error caused by wavelength deviation of the monochromator is smaller, which can improve detection accuracy and reduce measurement error.

[0024] Furthermore, by comparing the effects of hollow Fe3O4@MnO2 flower-shaped nanozymes and solid Fe3O4@MnO2 flower-shaped nanozymes on vitamin C detection, it was found that the hollow structure can broaden the detection range, lower the detection limit, and increase the maximum reaction rate V compared to the solid structure. max Larger and more affinity; comparing the effects of hollow nanozymes with different outer coating structures on vitamin C detection, it was found that only when the outer coating is flower-shaped and covers the hollow core, the detection range is the widest and the detection limit is the lowest. Therefore, the hollow flower-shaped structure of Fe3O4@MnO2 nanozymes is the key to achieving a wide detection range, high accuracy and low detection limit for vitamin C detection.

[0025] In another aspect, the present invention provides the use of Fe3O4@MnO2 nanozyme in vitamin C detection, wherein the Fe3O4@MnO2 nanozyme comprises a core and a coating outer layer, wherein the core is a Fe3O4 sphere and the coating outer layer is a MnO2 layer.

[0026] Preferably, the Fe3O4@MnO2 nanozyme is a hollow Fe3O4@MnO2 flower-shaped nanozyme.

[0027] In another aspect, the present invention provides the use of hollow Fe3O4@MnO2 flower-shaped nanozymes in expanding the detection range, improving detection accuracy, and lowering the detection limit for vitamin C detection. The hollow Fe3O4@MnO2 flower-shaped nanozyme includes a hollow core and a coating outer layer. The hollow core is a hollow Fe3O4 sphere; the coating outer layer is a MnO2 layer, and the coating outer layer coats the hollow core to form a flower-shaped structure.

[0028] In another aspect, the present invention provides a vitamin C detection kit, the kit comprising: Fe3O4@MnO2 nanozyme, TMB solution and NaAc-HAc buffer solution.

[0029] In some embodiments, the Fe3O4@MnO2 nanozyme is a hollow Fe3O4@MnO2 flower-shaped nanozyme.

[0030] In another aspect, the present invention provides a vitamin C detection kit for expanding the detection range of vitamin C detection, improving detection accuracy, and lowering the detection limit. The kit includes: hollow Fe3O4@MnO2 flower-shaped nanozyme, TMB solution, and NaAc-HAc buffer solution.

[0031] The present invention provides a hollow Fe3O4@MnO2 flower-shaped nanozyme, its preparation method, and its application in vitamin C content detection, which has the following beneficial effects: 1. Hollow Fe3O4@MnO2 flower-shaped nanozymes exhibit high stability and high catalytic activity; 2. Fe3O4@MnO2 can be used to detect vitamin C content. The operation is simple and time-saving, with a single sample detection time of only 5 minutes.

[0032] 3. Hollow Fe3O4@MnO2 flower-shaped nanozymes can broaden the linear range of vitamin C content detection to 0.568 ~ 567.8 µM; 4. Hollow Fe3O4@MnO2 flower-shaped nanozymes can reduce the limit of detection (LOD) for vitamin C content detection to 0.06 µM; 5. Hollow Fe3O4@MnO2 flower-shaped nanozymes have comparable accuracy to liquid chromatography in detecting vitamin C in test samples, but the experimental operation is simpler and the detection time is shorter than that of liquid chromatography. Attached Figure Description

[0033] Figure 1 These are scanning electron microscope (SEM) images of hollow Fe3O4 spheres and hollow Fe3O4@MnO2 flower-shaped nanozymes. (a) is a scanning electron microscope (SEM) image of hollow Fe3O4 spheres; (b) is a scanning electron microscope (SEM) image of hollow Fe3O4@MnO2 flower-shaped nanozymes. Figure 2 The image shows the energy dispersive X-ray spectroscopy (EDS) spectrum of the hollow Fe3O4@MnO2 flower-shaped nanoenzyme. (a) shows the elemental analysis region; (b) shows the distribution of iron (Fe); (c) shows the distribution of manganese (Mn); and (d) shows the distribution of oxygen (O). Figure 3The images show the X-ray diffraction (XRD) spectra of hollow Fe3O4 spheres and hollow Fe3O4@MnO2 flower-shaped nanozymes. Figure 4 The image shows the hysteresis loop of hollow Fe3O4@MnO2 flower-shaped nanoenzymes. Figure 5 The absorbance at 652 nm of the hollow Fe3O4@MnO2 flower-shaped nanozyme was measured in the same system under different temperatures, storage days, and recycling cycles. (a) is the absorbance at 652 nm measured at different temperatures; (b) is the absorbance at 652 nm measured at different storage days; and (c) is the absorbance at 652 nm measured at different recycling cycles. Figure 6 The absorption spectra of hollow Fe3O4@MnO2 flower-shaped nanozymes, hollow Fe3O4 spheres, and MnO2 nanosheets in the same system are measured at 500-700 nm. Figure 7 This is a linear correlation graph between the absorbance difference and vitamin C content when detecting vitamin C content using hollow Fe3O4@MnO2 flower-shaped nanozymes. Figure 8 The absorption spectrum and corresponding color of hollow Fe3O4@MnO2 flower-shaped nanozymes for detecting vitamin C content; Figure 9 This is a comparison chart of the detection values ​​and standard values ​​of hollow Fe3O4@MnO2 flower-shaped nanozymes when detecting citrus, VC chewable tablets, and NFC juice. (a) Detection object is citrus; (b) Detection object is VC chewable tablets; (c) Detection object is NFC juice. Figure 10 Optimized parameters for the activity of Fe3O4@MnO2 oxidases: (a) pH, (b) TMB concentration. Detailed Implementation

[0034] To describe the present invention more specifically, the technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are merely illustrative of how the present invention is implemented and do not limit the specific scope of the present invention. The scope of the present invention is defined in the claims.

[0035] Example 1: Preparation, structural characterization and catalytic performance of hollow Fe3O4@MnO2 flower-shaped nanozymes I. Preparation of hollow Fe3O4@MnO2 flower-shaped nanozymes 1. Preparation of hollow Fe3O4 spheres: 1.35 g of FeCl3·6H2O and 3.85 g of NH4Ac were dissolved in 70 mL of ethylene glycol and heated at 200 °C for 16 h. After washing, the spheres were dried at 60 °C for 12 h to obtain hollow Fe3O4 spheres.

[0036] 2. Preparation of hollow Fe3O4@MnO2 flower-shaped nanozyme: 0.3 g of the hollow Fe3O4 spheres synthesized in the first step were dispersed in 90 mL of deionized water under ultrasonication to form a uniform and stable suspension. Then, 0.75 g of KMnO4 was dissolved in the above suspension and stirred. Subsequently, 0.75 mL of hydrochloric acid was added dropwise and stirred continuously for 0.5 h. The mixture was heated at 120 °C for 6 h, washed, and dried at 60 °C for 12 h to obtain hollow Fe3O4@MnO2 flower-shaped nanozyme.

[0037] II. Structural Characterization 1. Observation using scanning electron microscopy (SEM) (1) Experimental methods A suitable amount of hollow Fe3O4@MnO2 flower-shaped nanoenzyme powder sample was taken and dispersed in anhydrous ethanol. The dispersion was ultrasonically treated for 5 minutes to obtain a uniform suspension, hereinafter referred to as the dispersion. A small amount of the suspension was pipetted onto a clean silicon wafer surface. After drying, the wafer was fixed to the SEM sample stage with conductive tape. The sample surface was then sputtered with gold using an ion sputtering instrument. The wafer was placed in the SEM sample chamber, and its microstructure was observed and photographed.

[0038] (2) Experimental results Experimental results are as follows Figure 1 As shown, Figure 1 Image (a) shows that the hollow Fe3O4 spheres have a diameter of approximately 300 nm, exhibiting a hollow structure; Figure 1 (b) shows that the hollow Fe3O4@MnO2 flower-shaped nanozyme has a diameter of about 1.5 µm and exhibits a flower-like structure, with MnO2 nanosheets growing on hollow Fe3O4 spheres.

[0039] 2. Energy-dispersive X-ray spectroscopy (EDS) (1) Experimental methods Sample preparation methods were the same as those used for scanning electron microscopy (SEM). After sample preparation, elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDS).

[0040] (2) Experimental results Experimental results are as follows Figure 2 As shown, Figure 2 (a) shows the area where elemental analysis was performed. Figure 2Figures 2(b) to 2(d) show the distribution of iron (Fe), manganese (Mn), and oxygen (O), respectively. Fe is mainly concentrated in the central region of the flower-shaped nanozyme, while Mn is mainly distributed on the periphery, and O is distributed throughout the entire nanozyme. This confirms the hollow core-shell structure of Fe3O4 and the flower-shaped structure formed by the growth of MnO2 nanosheets on hollow Fe3O4 spheres.

[0041] 3. X-ray diffraction (XRD) (1) Experimental methods The sample to be tested is thoroughly ground into a uniform fine powder in an agate mortar and filled into a grooved aluminum sample stage. The sample stage is then placed into an X-ray diffractometer for measurement.

[0042] (2) Experimental results Experimental results are as follows Figure 3 As shown, the XRD pattern of the hollow Fe3O4 spheres reveals multiple sharp characteristic diffraction peaks, indicating its high crystallinity. In addition to retaining the characteristic peaks of Fe3O4, the XRD pattern of the hollow Fe3O4@MnO2 flower-shaped nanozyme also shows new broad diffraction peaks. After comparison, these peaks were attributed to MnO2, confirming the successful synthesis of hollow Fe3O4@MnO2.

[0043] 4. Hysteresis loop (1) Experimental methods Hollow Fe3O4@MnO2 flower-shaped nanozyme powder was weighed and fixed onto the vibrating head of the sample rod of a vibrating sample magnetometer (VSM). The hysteresis loop diagram of the hollow Fe3O4@MnO2 flower-shaped nanozyme was measured and plotted using the vibrating sample magnetometer (VSM).

[0044] (2) Experimental results The hysteresis loop diagram of hollow Fe3O4@MnO2 flower-shaped nanoenzymes is shown below. Figure 4 As shown, the curve exhibits a typical "S" shape, with a saturation magnetization of approximately 33 emu / g and a residual magnetization close to zero, demonstrating that the nanozyme possesses excellent superparamagnetism and can respond rapidly under an applied magnetic field, facilitating separation and recovery.

[0045] III. Catalytic activity, stability, and recyclability (1) Experimental methods Take 100 µL of 100 µg / mL Fe3O4@MnO2 dispersion, 100 µL of 6 mM TMB solution, and 1000 µL of 0.1 M NaAc-HAc buffer solution at pH 4, mix them thoroughly, react at room temperature for 5 min, and measure the absorbance A at 652 nm.

[0046] (2) Experimental results Experimental results are as follows Figure 5 As shown, Figure 5 Images (a) to (c) show the absorbance at 652 nm of the hollow Fe3O4@MnO2 flower-shaped nanozyme in the same system under different temperatures, storage days, and recycling cycles. The catalytic activity of the nanozyme at different temperatures showed that the absorbance was around 1.0 within the temperature range of 20–80℃, indicating that the activity of the nanozyme is minimally affected by temperature and exhibits high stability. The absorbance of the nanozyme under different storage days showed that it maintained high catalytic activity for up to 30 days, indicating good long-term storage stability. The absorbance of the system under different recycling cycles showed that after magnetic separation and recycling, and reused 5 times, the catalytic activity did not significantly decrease, confirming the excellent stability and reusability of the nanozyme. This is because its excellent superparamagnetism allows for rapid recovery, and the recovered nanozyme still maintains high catalytic activity.

[0047] like Figure 6 As shown, the absorption spectra of hollow Fe3O4@MnO2 flower-shaped nanozymes, hollow Fe3O4 spheres, and MnO2 nanosheets were measured in the same system within the range of 500–700 nm. The results showed that the absorbance of hollow Fe3O4@MnO2 flower-shaped nanozymes at 652 nm was much higher than that of hollow Fe3O4 spheres and MnO2 nanosheets, indicating that the catalytic efficiency of hollow Fe3O4@MnO2 flower-shaped nanozymes was significantly better than that of hollow Fe3O4 spheres and MnO2 nanosheets.

[0048] Example 2: Application of hollow Fe3O4@MnO2 flower-shaped nanozymes in the detection of vitamin C content Plotting the standard curve: Take 100 µL of a 100 µg / mL Fe3O4@MnO2 dispersion, 100 µL of a 6 mM TMB solution, and 1000 µL of a 0.1 M NaAc-HAc buffer solution (pH 4). Mix thoroughly and react at room temperature. Then add 100 µL of vitamin C standards at concentrations of 0.568 µM (0.1 µg / mL), 5.68 µM (1 µg / mL), 56.8 µM (10 µg / mL), 284 µM (50 µg / mL), and 568 µM (100 µg / mL), respectively. Measure the absorbance difference ΔA (the difference in absorbance at 652 nm between the blank system and the system with added vitamin C standards) at 652 nm. A linear positive correlation exists between the vitamin C content and ΔA. Figure 7 As shown, the linear equation is y = 0.0134x + 0.0908, R02 =0.999. This nanozyme exhibits a low limit of detection (LOD) and a wide detection range for vitamin C, ranging from 0.568 to 567.8 µM, with an LOD as low as 0.06 µM. For example... Figure 8 As shown, the absorption spectrum of hollow Fe3O4@MnO2 flower-shaped nanozyme for detecting vitamin C standards and the corresponding colors in the system clearly indicate that the absorbance at 652 nm decreases with increasing vitamin C content. Simultaneously, vitamin C reacts with the blue oxidation product (TMB)... OX As the vitamin C content increases, the color of the solution gradually changes from dark blue to light blue, and eventually to colorless.

[0049] Determination of Vitamin C content in the test sample: Take 100 µl of Fe3O4@MnO2 dispersion with a concentration of 100 µg / mL, 100 µl of TMB solution with a concentration of 6 mM, and 1000 µL of NaAc-HAc buffer solution with a concentration of 0.1 M and a pH of 4. Mix them thoroughly and react at room temperature. Add the test sample and measure the absorbance difference ΔA at 652 nm. Calculate the vitamin C content in the test sample using a standard curve.

[0050] Citrus fruits, Centrum Vitamin C chewable tablets (hereinafter referred to as VC chewable tablets), and Nongfu Spring NFC fresh-squeezed orange juice (hereinafter referred to as NFC juice) purchased from the market were selected as test samples. The vitamin C content was detected using the method described in this application, and liquid chromatography was used as a control. The liquid chromatography method was performed according to the experimental conditions and procedures in Method I of GB 5009.86-2016 National Food Safety Standard for the Determination of Ascorbic Acid in Food. The experimental results are as follows: Figure 9 As shown, Figure 9 Images (a) to (c) show the comparison between the detection values ​​obtained by the above method and the standard values ​​measured by liquid chromatography when using hollow Fe3O4@MnO2 flower-shaped nanozymes to detect citrus fruits, vitamin C chewable tablets, and NFC juice. For these three analytes containing vitamin C, the nanozymes based on this invention achieved a high degree of agreement between the vitamin C content determination values ​​and the standard values ​​measured by liquid chromatography, indicating that the method has excellent detection accuracy.

[0051] Furthermore, the accuracy of the method was further verified using a spiked recovery method. Specifically, three samples of citrus juice (juiced from commercially purchased citrus fruits) with known vitamin C content were selected. Vitamin C standard solutions of 10, 15, and 20 µg / mL were added respectively. The spiked samples were then analyzed and the recovery rate was calculated according to the method described in Example 2. The experimental results are shown in Table 1. The p-value between the UV method and the chromatographic method was greater than 0.05, indicating no statistically significant difference between the two methods. These results fully verify the accuracy and reliability of hollow Fe3O4@MnO2 flower-shaped nanozymes for vitamin C content detection.

[0052] Table 1. Recovery analysis of vitamin C in citrus juice using ultraviolet light and chromatography, with 3 replicates (n)

[0053] Example 3: Effect of hollow structure on catalytic activity The hollow Fe3O4@MnO2 flower-shaped nanozyme was prepared using the method described in Example 1. The heating conditions for preparing the solid flower-shaped Fe3O4@MnO2 spheres were changed to 200 °C for 6 h.

[0054] The effects of hollow and solid structures on vitamin C detection were compared using the method described in Example 2. The results showed that, compared to solid nanozymes, hollow Fe3O4@MnO2 flower-shaped nanozymes exhibited a wider detection range for vitamin C (0.568–567.8 μM), a lower limit of detection (LOD) of 0.06 μM, and a higher maximum reaction rate V0. max Larger, at 12.35 × 10 -8 M / s, Michael constant (K) m The lower value indicates a stronger affinity for the substrate, suggesting that hollow Fe3O4@MnO2 nanozymes have a significant positive effect on broadening the application range of vitamin C detection and improving detection accuracy.

[0055] Table 2 Comparison of Vitamin C Content Detection Performance between Hollow and Solid Structure Materials

[0056] Example 4: Effect of outer coating morphology on catalytic activity Based on the hollow Fe3O4 spheres prepared in Example 1, an outer coating layer, i.e. a shell layer, was prepared. By heating at 100 °C for 1 h, 100 °C for 6 h, and 120 °C for 6 h, mushroom-shaped, honeycomb-shaped, and flower-shaped hollow Fe3O4@MnO2 nanozymes were obtained, and the specific groupings are shown in Table 3.

[0057] Table 3 Selection of outer coating structure

[0058] Using the vitamin C content detection method described in Example 2, the effect of nanozymes with different outer coating structures on vitamin C content detection was studied. The experimental results are shown in Table 4. When the outer coating structure is flower-shaped, the detection range for vitamin C content detection is the widest, ranging from 0.568 to 567.8 µM, and the limit of detection (LOD) is the lowest, at 0.06 µM, compared to other outer coating structures. This indicates that the flower-shaped Fe3O4@MnO2 nanozyme has a significant positive effect on broadening the application range of vitamin C detection and improving detection accuracy.

[0059] Table 4. Detection results of vitamin C content of nanozymes with different outer coating structures.

[0060] Example 5: Effects of buffer solution pH and substrate TMB concentration on oxidase-like activity Prepare 0.1 M NaAc-HAc buffer solutions and adjust the pH to 3.0–7.0. Take 1000 µL of each pH buffer solution, add 100 µL of hollow Fe3O4@MnO2 flower-shaped nanozyme dispersion and 100 µL of TMB solution (6 mM), respectively, mix well, and react at room temperature for 5 minutes. Measure the absorbance of each system at 652 nm. Figure 10 As shown in (a), the absorbance at 652 nm first increases and then decreases in the pH range of 3.0 to 7.0, reaching its maximum value at pH 4, indicating that the nanozyme has the best oxidase-like catalytic activity at pH 4.

[0061] Prepare TMB solutions with concentrations ranging from 0.1 to 20 mM. Take 1000 µL of the buffer solution, add 100 µL of hollow Fe3O4@MnO2 flower-shaped nanozyme dispersion and 100 µL of TMB solution respectively, mix thoroughly, and react at room temperature for 5 minutes. Measure the absorbance of each system at 652 nm. Figure 10 As shown in (b), the absorbance is high at a TMB concentration of 6 mM, and tends to stabilize with increasing TMB concentration, indicating that the effect of TMB concentration on improving catalytic efficiency has reached saturation. Therefore, a TMB concentration of 6 mM is preferred, at which a balance can be achieved between sufficient substrate and reduced background interference.

[0062] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for detecting vitamin C using hollow Fe3O4@MnO2 flower-shaped nanozymes, characterized in that, The method includes the following steps: (1) Preparation of hollow Fe3O4@MnO2 flower-shaped nanozyme: 1.35 g of FeCl3·6H2O and 3.85 g of NH4Ac were dissolved in 70 mL of ethylene glycol and heated at 200 °C for 16 h. After washing, the nanozyme was dried at 60 °C for 12 h to obtain hollow Fe3O4 spheres. 0.3 g of hollow Fe3O4 spheres were dispersed in 90 mL of deionized water under ultrasonic treatment to form a uniform and stable suspension. Then, 0.75 g of KMnO4 was dissolved in the above suspension and stirred. Subsequently, 0.75 mL of hydrochloric acid was added dropwise and stirred continuously for 0.5 h. The nanozyme was heated at 120 °C for 6 h and dried at 60 °C for 12 h to obtain hollow Fe3O4@MnO2 flower-shaped nanozyme. (2) Construction of standard curve: Hollow Fe3O4@MnO2 flower-shaped nanoenzyme powder was dispersed in anhydrous ethanol and ultrasonically dispersed for 5 minutes to obtain a uniform suspension. A 100 µg / mL Fe3O4@MnO2 dispersion was prepared. 100 µL of the 100 µg / mL Fe3O4@MnO2 dispersion, 100 µL of 6 mM TMB solution, and 1000 µL of 0.1 M NaAc-HAc buffer solution with pH 4 were mixed evenly and reacted at room temperature. Then, 100 µL of vitamin C standard at concentrations of 0.568 µM (0.1 µg / mL), 5.68 µM (1 µg / mL), 56.8 µM (10 µg / mL), 284 µM (50 µg / mL), and 568 µM (100 µg / mL) were added respectively. The mixture was then heated at 652 °C. The absorbance difference ΔA between the blank system and the system containing vitamin C standard was measured at 652 nm. A linear positive correlation was observed between the vitamin C content and ΔA, with the linear equation y = 0.0134x + 0.0908, R0. 2 =0.999; (3) Take 100 µL of 100 µg / mL Fe3O4@MnO2 dispersion, 100 µL of 6 mM TMB solution, and 1000 µL of 0.1 M NaAc-HAc buffer solution with pH 4. Mix them evenly, react at room temperature, add the test sample, measure the absorbance difference at 652 nm, and calculate the vitamin C content in the test sample through the standard curve.

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