Method for detecting vitamin c based on oxidase activity nanosize enzyme

Abstract

This invention provides a method for detecting vitamin C based on oxidase-active nanozymes. The method includes: preparing a first mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer, and the sample to be tested; and a second mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer, and vitamin C at different concentration gradients; reacting the first and second mixtures under the same predetermined reaction conditions; measuring the absorbance of the products after the reaction at predetermined wavelengths, corresponding to absorbance values ​​A1 and A0, respectively; obtaining a linear regression equation based on A0 and vitamin C at different concentration gradients; and determining the vitamin C content in the sample to be tested based on the linear regression equation and A1. The provided method is rapid and has extremely high sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, specifically relating to a method for detecting vitamin C based on nanozymes with oxidase activity. Background Technology

[0002] Vitamin C (VC) is an important water-soluble vitamin widely found in fresh fruits and vegetables, and is essential for human health. It participates in various biochemical and physiological processes, including the absorption of metal ions, collagen synthesis, and maintaining normal blood vessel and adrenal gland secretion. Furthermore, VC's strong free radical scavenging ability helps maintain the body's redox balance. Insufficient vitamin C intake can lead to serious health problems such as scurvy, cancer, Alzheimer's disease, Parkinson's disease, and cardiovascular disease. Therefore, the vitamin C content in fruits and vegetables is an important indicator of their quality. To accurately assess the vitamin C content in fruits and vegetables, various sensitive methods have been developed, including chromatography, titration, electrochemistry, capillary electrophoresis, enzymatic methods, fluorescence analysis, and ultraviolet light methods. These methods offer high reproducibility and selectivity, enabling the analysis of trace amounts of vitamin C. Among these, spectroscopic methods have received increasing attention due to their low cost and ease of operation. Spectroscopic colorimetric sensors, due to their ease of operation and visual inspection advantages, have become important colorimetric tools, facilitating the assessment of vitamin C content.

[0003] Enzyme-dependent colorimetric assays are widely used for the detection of various targets due to their high selectivity and ease of operation. However, the use of natural enzymes is limited by various factors, including environmental conditions and the enzyme's molecular structure, which can lead to enzyme inactivation. Furthermore, the preparation and purification of natural enzymes are often complex and costly, severely limiting their feasibility for large-scale applications. Therefore, enzyme mimics with similar activities have attracted widespread attention as an alternative. In recent years, studies have found that various nanomaterials (such as noble metal nanoparticles, metal oxides, transition metal carbides, and metal-organic frameworks) possess intrinsic enzyme-like activity and have been applied to the colorimetric detection of various target molecules. In particular, sensors based on oxidase-like nanozymes have become a considerable colorimetric platform. Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems in related technologies. This invention provides a highly efficient and accurate oxidase-driven method for the detection of vitamin C. This invention provides a MnO2@MIP nanozyme that can generate reactive oxygen species (ROS) to catalyze the conversion of colorless 3,3',5,5'-tetramethylbenzidine (TMB) into a blue oxidation product (oxTMB). In the presence of vitamin C (VC), VC can fill the specifically imprinted cavity of the MnO2@MIP, hindering the contact between TMB and the catalytic center MnO2, thus inhibiting the oxidation of TMB. Simultaneously, VC, as an antioxidant, can scavenge ROS, thereby causing the blue oxTMB to fade. In summary, a method for the adsorption and detection of vitamin C based on oxidase-active nanozyme adsorption has been developed through colorimetric reaction, which can be used to accurately determine the vitamin C content.

[0005] Specifically, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for detecting vitamin C based on an oxidase-active nanozyme, comprising:

[0007] Prepare a first mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested, and a second mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and vitamin C at different concentration gradients;

[0008] The first mixture and the second mixture react under the same predetermined reaction conditions, and the absorbance of the reaction products at a predetermined wavelength is measured, corresponding to absorbance values ​​A1 and A0, respectively.

[0009] A linear regression equation is obtained based on the absorbance value A0 and vitamin C at different concentration gradients. Based on the linear regression equation and the absorbance value A1, the vitamin C content in the sample to be tested is determined.

[0010] The MnO2@MIP nanozyme was synthesized by the following method:

[0011] The MnO2 nanosheets, N-n-octyl-D-glucosamine, and vitamin C were mixed and subjected to a first reaction, followed by a second reaction with ethylene glycol dimethacrylate. The vitamin C was then washed away to obtain the MnO2@MIP nanozyme.

[0012] According to embodiments of the present invention, the method for detecting vitamin C based on oxidase-active nanozymes provided above may further include the following technical features:

[0013] According to an embodiment of the present invention, the predetermined wavelength is 640-660 nanometers, preferably 650 nanometers.

[0014] According to an embodiment of the present invention, the predetermined reaction conditions are a reaction at a temperature of 30 to 40 degrees Celsius for 5 to 10 minutes.

[0015] According to an embodiment of the present invention, the pH value of the HAc-NaAc buffer solution is 4.8-5.2; the concentration of the TMB solution is 5-10.0 mmol / L; the concentration of the MnO2@MIP nanozyme is 40-50 μg / mL; and the total volume of the mixed solution is 200.0 μL.

[0016] According to an embodiment of the present invention, the linear regression equation is A = -0.01185C. VC +1.87528.

[0017] According to an embodiment of the present invention, the MnO2@MIP nanozyme is prepared by the following method:

[0018] MnO2 nanosheets were obtained based on the mixed reaction of tetramethylammonium hydroxide, hydrogen peroxide and manganese chloride;

[0019] MnO2 nanosheets, N-n-octyl-D-glucosamine, and vitamin C were mixed and subjected to a first reaction at 20-30°C. Then, they were mixed with ethylene glycol dimethacrylate and subjected to a second reaction at 65-75°C. The vitamin C was removed by washing to obtain MnO2@MIP nanozyme.

[0020] According to an embodiment of the present invention, the MnO2@MIP nanozyme is prepared by the following method:

[0021] S1: Tetramethylammonium hydroxide solution, H2O2 solution and deionized water are mixed, then MnCl2·4H2O solution is added, the reaction is carried out, and the mixture is washed to obtain bulk manganese dioxide. Then the bulk manganese dioxide is dissolved in deionized water and the resulting solution is freeze-dried to obtain MnO2 nanosheets.

[0022] S2: The prepared MnO2 nanosheets, N-n-octyl-D-glucosamine and vitamin C were added to dichloromethane and stirred at 20-30℃ for 6-8 hours. Then ethylene glycol dimethacrylate was added and stirred at 65-75℃ for 11-13 hours to obtain an intermediate product. After washing and drying, the MnO2@MIP nanozyme was obtained.

[0023] According to an embodiment of the present invention, the sample to be tested is a protein sample. The provided method can be used to detect the vitamin C content in proteins. The protein sample contains at least one of the following: urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, and Cu. 2+ Ca 2+ Mg 2+ Al 3+ K + Na + The method provided is unaffected by these substances.

[0024] A second aspect of the present invention provides a method for detecting vitamin C in proteins based on oxidase-active nanozymes, comprising:

[0025] Prepare a first mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested, and a second mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and vitamin C at different concentration gradients;

[0026] The first mixture and the second mixture were reacted at a temperature of 30 to 40 degrees Celsius for 5 to 10 minutes, and the absorbance of the reaction product at a wavelength of 640 to 660 nanometers was measured, corresponding to absorbance values ​​A1 and A0, respectively.

[0027] A linear regression equation is obtained based on the absorbance value A0 and vitamin C at different concentration gradients. Based on the linear regression equation and the absorbance value A1, the vitamin C content in the sample to be tested is determined.

[0028] The MnO2@MIP nanozyme was synthesized by the following method:

[0029] The first reaction was carried out by mixing MnO2 nanosheets, N-n-octyl-D-glucosamine and vitamin C, followed by a second reaction by mixing with ethylene glycol dimethacrylate. The vitamin C was then washed away to obtain MnO2@MIP nanozyme.

[0030] A third aspect of the present invention provides a method for detecting vitamin C in proteins based on oxidase-active nanozymes, comprising:

[0031] MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested were mixed, and vitamin C of different concentration gradients was added to obtain mixed solutions.

[0032] The mixed solution was reacted at a temperature of 30–40 degrees Celsius for 5–10 minutes, and the absorbance of the reaction product at a wavelength of 640–660 nanometers was measured.

[0033] The vitamin C content in the sample to be tested was determined based on the absorbance values ​​of different mixed solutions.

[0034] The MnO2@MIP nanozyme was synthesized by the following method:

[0035] MnO2 nanosheets were obtained by reacting a mixture of tetramethylammonium hydroxide solution, hydrogen peroxide solution, and manganese chloride solution.

[0036] The MnO2 nanosheets, N-n-octyl-D-glucosamine, and vitamin C were reacted, and then reacted with ethylene glycol dimethacrylate to obtain MnO2@MIP nanozymes.

[0037] A fourth aspect of the present invention provides a MnO2@MIP nanozyme for colorimetric detection of vitamin C, which is prepared by the following method:

[0038] The first reaction was carried out by mixing MnO2 nanosheets, N-n-octyl-D-glucosamine and vitamin C, followed by a second reaction by mixing with ethylene glycol dimethacrylate. The vitamin C was then washed away to obtain MnO2@MIP nanozyme.

[0039] A fifth aspect of the present invention provides a vitamin C assay kit, comprising: MnO2@MIP nanozyme, HAc-NaAc buffer, and TMB solution. The vitamin C assay kit may also include vitamin C standards as needed.

[0040] The beneficial effects achieved by this invention are at least as follows:

[0041] (1) The method of the present invention can detect vitamin C in a shorter time, and the color change of the test reagent can be observed by the naked eye in only 5.0 minutes.

[0042] (2) The detection method provided by the present invention does not add H2O2, which effectively avoids false positive output caused by directly adding unstable H2O2.

[0043] (3) The MnO2@MIP nanozyme used in this invention exhibits outstanding performance in terms of selectivity and anti-interference. Its highly adaptable three-dimensional cavity for VC molecules excludes representative substrates in proteins and VC structural analogs such as urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, and Cu. 2+ Ca 2+ Mg 2+ Al 3+ K + Na + It is resistant to interference from factors such as [list of factors], exhibiting good selectivity and anti-interference capabilities.

[0044] (4) The detection method adopted in this invention has extremely high sensitivity, with a detection limit (LOD) as low as 0.253 μM. Attached Figure Description

[0045] Figure 1 TEM image of MnO2@MIP provided according to an embodiment of the present invention.

[0046] Figure 2 Figure A shows the characterization of MnO2@MIP according to an embodiment of the present invention. Figure B shows the hydrodynamics of MnO2@MIP, Figure C shows the Zeta potential of MnO2@MIP, and Figure D shows the XPS full spectrum of MnO2@MIP.

[0047] Figure 3 The XPS high-resolution elemental spectrum of MnO2@MIP provided according to an embodiment of the present invention includes C, N, O, and Mn.

[0048] Figure 4 Optimization of conditions for detecting VC in the MnO2@MIP+TMB system provided in the embodiments of the present invention includes pH value and temperature.

[0049] Figure 5 To verify the feasibility of detecting VC using the MnO2@MIP+TMB system provided in the embodiments of the present invention, different groups were selected; the ultraviolet absorption spectra and corresponding solution color illustrations of (1) VC, (2) TMB, (3) MnO2@MIP, (4) MnO2@MIP+TMB, and (5) MnO2@MIP+TMB+VC were obtained.

[0050] Figure 6 The UV absorption spectra and standard curves of the reverse system containing different concentrations of VC standards are provided according to embodiments of the present invention.

[0051] Figure 7 The selectivity and anti-interference performance of the MnO2@MIP+TMB detection system provided according to an embodiment of the present invention are tested. Figures A1-15 show the results for blank, VC, urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, and Cu, respectively. 2+ Ca 2+ Mg 2+ Al 3+ K + Na + Figure B shows 1-14, representing VC+ blank, urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, and Cu, respectively. 2+ Ca 2+ Mg 2+ Al3+ K + Na + . Detailed Implementation

[0052] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0053] This invention provides a method for detecting vitamin C content in proteins using a nanozyme based on oxidase activity, which has significant biological and medical implications. First, vitamin C (ascorbic acid) plays several crucial roles in the human body, including promoting collagen synthesis, enhancing immunity, acting as an antioxidant, and participating in the regulation of various enzyme activities. Vitamin C deficiency can lead to serious health problems such as scurvy, weakened immunity, and poor wound healing. Detecting the vitamin C content in proteins can help assess the nutritional value of food or biological samples. Since vitamin C cannot be synthesized in the human body and must be obtained through diet, its content is one of the important indicators for evaluating the nutritional value of food. Furthermore, the detection of vitamin C is also crucial for ensuring the quality of food and pharmaceuticals. Additionally, vitamin C, as a cofactor for various hydroxylases, is essential for collagen synthesis, and collagen is the most abundant protein in the human body, playing a key role in the structural integrity of tissues throughout the body. Therefore, detecting the vitamin C content in proteins helps to understand its functional status in organisms, especially in the study of collagen-related diseases. Detecting the vitamin C content in proteins not only helps assess nutrient intake and food quality, but also helps understand the physiological functions of vitamin C in organisms and the potential health effects of deficiency or excess.

[0054] This invention provides a MnO2@MIP nanozyme, which is synthesized by the following method:

[0055] The first reaction was carried out by mixing MnO2 nanosheets, N-n-octyl-D-glucosamine and vitamin C, followed by a second reaction by mixing with ethylene glycol dimethacrylate. The vitamin C was then washed away to obtain MnO2@MIP nanozyme.

[0056] The provided method for synthesizing nanozymes involves adding vitamin C as a template, encapsulating it around the nanozyme, and then eluting to remove the vitamin C, leaving a specific cavity for vitamin C. This cavity is specific for vitamin C, thus achieving the purpose of detecting and adsorbing vitamin C. Compared with other nanozymes, it has a shorter detection time, better selectivity, and is unaffected by various interferences.

[0057] According to a specific embodiment, the provided method for detecting vitamin C based on oxidase-active nanozymes includes the following steps:

[0058] Step 1: Measure the absorbance of the test solution of vitamin C standards at different concentration gradients at 650 nm:

[0059] Under room temperature conditions, MnO2@MIP nanozyme, TMB, and vitamin C at different concentration gradients were added to HAc-NaAc buffer solution for reaction. The mixture was thoroughly stirred, and the reaction was allowed to continue for 5.0 min, after which the solution was transferred to a cuvette. The absorbance at 650 nm was then measured using a UV spectrophotometer and recorded as A0.

[0060] Step 2: Construct the linear regression equation for the standard sample

[0061] An ultraviolet regression equation, A0 = XC, was constructed using the absorbance values ​​measured from vitamin C standards at different concentration gradients and the concentrations of the standards. VC +y, where C VC For standard concentration;

[0062] Step 3: Take the sample to be tested, repeat step 1 to obtain the absorbance value A1, and substitute it into the corresponding linear equation to obtain the concentration of vitamin C in the sample to be tested.

[0063] In step 1, the pH of the HAc-NaAc buffer solution is 4.8-5.2; the concentration of the TMB solution is 10.0 mmol / L; the concentration of the MnO2@MIP nanozyme is 50.0 μg / mL; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 35.0℃; and the reaction time is 5.0 min.

[0064] According to a specific implementation method, the linear regression equation is A0 = -0.01185C. VC +1.87528;

[0065] According to a specific embodiment, the preparation method of the MnO2@MIP nanozyme includes the following steps:

[0066] S1: Tetramethylammonium hydroxide solution (TMAH, 25 wt%) and H2O2 solution (3 wt%) were thoroughly mixed with deionized water, and then MnCl2·4H2O solution was added. The mixture was stirred overnight in the open air. Then, it was washed three times with ultrapure water and methanol, and after centrifugation, purification, and drying, lumpy manganese dioxide was collected. The lumpy MnO2 was then added to deionized water and sonicated for 1-3 hours. The resulting solution was freeze-dried to obtain MnO2 nanosheets.

[0067] S2: The prepared MnO2 nanosheets, N-n-octyl-D-glucosamine and vitamin C were added to dichloromethane (DCM) and stirred at 20-30℃ for 6-8 h. Then ethylene glycol dimethacrylate was added and stirred at 65-75℃ for 11-13 h to obtain an intermediate product. The vitamin C in the intermediate product was removed by washing, filtered and dried at 65-75℃ to obtain the molecularly imprinted polymer MnO2@MIP for adsorbing vitamin C.

[0068] The technical solution of the present invention will be described below through specific embodiments. It should be noted that the following embodiments are provided to better understand the present invention, and not to limit the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all purchased from conventional biochemical reagent stores.

[0069] Reagents used in the embodiments of this invention:

[0070] The raw materials used in this invention are as follows: tetramethylammonium hydroxide solution (TMAH, 25 wt%) and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; manganese chloride tetrahydrate, N-n-octyl-D-glucosamine, ethylene glycol dimethacrylate, and vitamin C (ascorbic acid) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and acetate-sodium acetate buffer (HAc-NaAc) buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd. All chemicals were purchased directly from the suppliers without further purification; and ultrapure water was used throughout the experiments.

[0071] Unless otherwise specified, all reagents or materials described in the following examples are commercially available.

[0072] Example 1

[0073] Example 1 describes the preparation and synthesis of MnO2@MIP nanozymes using the following method:

[0074] (1) Tetramethylammonium hydroxide solution (TMAH, 25 wt%) and H2O2 solution (3 wt%) were thoroughly mixed with deionized water, and then MnCl2·4H2O solution was added. The mixture was stirred overnight in the open air. Then, it was washed three times with ultrapure water and methanol, and collected as lumpy manganese dioxide after centrifugation, purification and drying. The lumpy MnO2 was then added to deionized water and sonicated for 2 h. The resulting solution was freeze-dried to obtain MnO2 nanosheets.

[0075] (2) The prepared MnO2 nanosheets, N-n-octyl-D-glucosamine and vitamin C were added to dichloromethane (DCM) and stirred at 25°C for h. Then ethylene glycol dimethacrylate was added and stirred at 70°C for 12 h. The product was washed with methanol for 2 hours to remove vitamin C from the intermediate product. After filtration, it was dried at 70°C to obtain MnO2@MIP nanozyme.

[0076] Example 2

[0077] Example 2 optimized the conditions for vitamin C detection using the MnO2@MIP nanozyme prepared in Example 1, especially by studying experimental parameters such as pH and temperature in the detection system in detail.

[0078] The experiment first investigated the performance of the system within a pH range of 3.5-5.5, and HAc-NaAc buffer was selected as the reaction buffer. The results are as follows... Figure 4 As shown in Figure A, the results indicate that when the pH value is in the range of 3.5 to 5.0, the absorbance value of the reaction system at 650 nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 5.0, the absorbance value decreases with increasing pH value. Therefore, 5.0 was selected as the optimal pH value for the system.

[0079] The performance of this system was then investigated in the temperature range of 25.0–45.0 °C, and the results are as follows: Figure 4 As shown in Figure B. Experimental results show that when the temperature ranges from 25.0℃ to 35.0℃, the absorbance of the reaction system at 650nm increases with increasing reaction temperature. When the temperature exceeds 35.0℃ and increases to 45.0℃, the absorbance decreases with increasing temperature. Therefore, 35.0℃ was chosen as the optimal temperature for the system.

[0080] Example 3

[0081] Example 3 verified the feasibility of using MnO2@MIP nanozymes to detect vitamin C, including:

[0082] Using TMB as the colorimetric reagent and HAc-NaAc as the buffer, the following five reaction groups were performed. The groups are numbered as follows:

[0083] (1) VC, (2) TMB, (3) MnO2@MIP, (4) MnO2@MIP+TMB, (5) MnO2@MIP+TMB+VC. After the reaction is complete, transfer the mixture to a cuvette and measure the absorbance at 650 nm using a UV spectrophotometer. Compare the values ​​of each cuvette.

[0084] See results Figure 5This indicates that vitamin C can inhibit its oxidase activity, causing a significant change in absorbance. The experiment demonstrates the feasibility of the detection system proposed in this invention.

[0085] In the above verification steps, the concentration of MnO2@MIP nanozyme was 50.0 mg / mL; the concentration of TMB was 10.0 mmol / L; the concentration of VC was 120 mM; the pH of the added HAc-NaAc buffer was 4.8-5.2; the total volume of the mixed solution was 200.0 mL; the reaction temperature was 35.0 °C; and the mixing and reaction time was 5.0 min.

[0086] Example 4

[0087] Example 4 utilizes MnO2@MIP nanozymes to detect vitamin C standards at different concentrations, including:

[0088] Different concentrations of VC solutions (including 0.0, 10.0, 20.0, 40.0, 60.0, 80.0, 100.0, 120.0 and 140.0 mM) were reacted with 50.0 μg / mL MnO2@MIP nanozyme and 10.0 mmol / L TMB solution in HAc-NaAc buffer (pH = 4.8-5.2) at 35.0 °C for 5.0 min.

[0089] After the reaction was complete, the reaction solution was transferred to a cuvette, and the absorbance at 650 nm was measured using a UV spectrophotometer. The absorbance at 650 nm decreased with increasing VC concentration. Therefore, a linear equation was fitted using Origin software with absorbance A as the ordinate and VC concentration as the abscissa.

[0090] The result is as follows Figure 6 As shown, the concentration of VC and absorbance A exhibit a linear relationship in the range of 0-120.0 mM, with the absorbance regression equation being A = -0.01185C. VC The correlation coefficient was +1.87528, the detection limit was 0.99322, and the detection limit was 0.253 μM. Furthermore, as the concentration of vitamin C increased, the solution exhibited a color gradient (from dark blue to light blue), which could be observed visually, enabling the detection of vitamin C concentration.

[0091] Example 5

[0092] Example 5 tested the selectivity and anti-interference ability of the method provided by the present invention for detecting vitamin C. Representative contents were used as proof of concept, including 1-15 corresponding to blank, vitamin C, urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, and Cu, respectively. 2+ Ca 2+ Mg 2+ Al3+ K + Na + .include:

[0093] At room temperature, MnO2@MIP nanozyme and TMB were reacted with the above substances in HAc-NaAc buffer solution; after thorough mixing, the mixture was reacted for 5.0 min and then transferred to a cuvette. The absorbance value (A) at 650 nm was measured. The results are as follows. Figure 7 As shown in Figure A, only the absorbance value A of VC showed a significant decrease, indicating that the detection system has good selectivity for VC. The effects of VC coexisting with these substances on the reaction system were also investigated, and the results are as follows. Figure 7 As shown in Figure B, the absorbance values ​​at 650 nm of each coexisting system did not change significantly, indicating that the constructed detection system has good anti-interference ability.

[0094] The concentration of the added MnO2@MIP nanozyme was 50.0 μg / mL; the concentration of TMB was 10.0 mmol / L; the concentration of the selective validation substance was 120.0 mM; the pH of the added HAc-NaAc buffer was 4.8-5.2; the total mixed solution was 200.0 mL; the reaction temperature was 35.0 ℃; and the mixing and reaction time was 5.0 min.

[0095] Example 6

[0096] Example 6 uses the method provided by the present invention to detect vitamin C in a real sample.

[0097] Using soy protein (randomly purchased from a large supermarket in Hefei, China, without purification) as a representative, 1g of soy protein was added to 10.0mL of deionized water and the pH was adjusted to 5.0. Then, 10.0, 60.0, and 120.0mM vitamin C were added respectively. At room temperature, MnO2@MIP and TMB were reacted with the above mixed solutions in HAc-NaAc buffer; after mixing thoroughly, the reaction was allowed to proceed for 5.0min, and then transferred to cuvettes. The absorbance value A at 650nm was measured. Substituting the obtained absorbance value A into the corresponding linear equation, the vitamin C results measured in colorimetric detection mode are shown in Table 1.

[0098] In addition, pea protein (randomly purchased from a large supermarket in Hefei, China, without purification) was selected as a representative sample. 1g of pea protein was added to 10.0mL of deionized water, and the pH was adjusted to 5.0. Then, 10.0, 60.0, and 120.0mM vitamin C were added respectively. At room temperature, MnO2@MIP and TMB were reacted with the above mixed solutions in HAc-NaAc buffer; after thorough mixing, the reaction was allowed to proceed for 5.0min, and then transferred to cuvettes. The absorbance value A at 650nm was measured. Substituting the obtained absorbance value A into the corresponding linear equation, the vitamin C results measured in colorimetric detection mode are shown in Table 1.

[0099] We also selected chickpea protein (randomly purchased from a large supermarket in Hefei, China, without purification) as a representative sample. 1g of chickpea protein was added to 10.0mL of deionized water and the pH was adjusted to 5.0. Then, 10.0, 60.0, and 120.0mM vitamin C were added respectively. At room temperature, MnO2@MIP and TMB were reacted with the above mixed solutions in HAc-NaAc buffer; after thorough mixing, the reaction was allowed to proceed for 5.0 min, and then transferred to cuvettes. The absorbance value A at 650nm was measured. Substituting the obtained absorbance value A into the corresponding linear equation, the vitamin C results measured in colorimetric detection mode are shown in Table 1.

[0100] Table 1 shows the detection of VC in real samples using the MnO2@MIP+TMB system.

[0101]

[0102] As can be seen from the results in Table 1 above, the reaction system can be applied to the detection of actual samples, and the recovery rate is between 98% and 105%, with a relative deviation of less than 3.00%.

[0103] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," and "detailed description" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0104] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for detecting vitamin C based on nanozymes with oxidase activity, characterized in that, include: Prepare a first mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested, and a second mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and vitamin C at different concentration gradients; The first mixture and the second mixture react under the same predetermined reaction conditions, and the absorbance of the reaction products at a predetermined wavelength is measured, corresponding to absorbance values ​​A1 and A0, respectively. A linear regression equation is obtained based on the absorbance value A0 and vitamin C at different concentration gradients. Based on the linear regression equation and the absorbance value A1, the vitamin C content in the sample to be tested is determined. The MnO2@MIP nanozyme was synthesized by the following method: The MnO2 nanosheets, N-n-octyl-D-glucosamine, and vitamin C were mixed and subjected to a first reaction, followed by a second reaction with ethylene glycol dimethacrylate. The vitamin C was then washed away to obtain the MnO2@MIP nanozyme.

2. The method according to claim 1, characterized in that, The predetermined wavelength is 640~660 nanometers.

3. The method according to claim 1, characterized in that, The predetermined wavelength is 650 nanometers.

4. The method according to claim 1, characterized in that, The predetermined reaction conditions are to react at a temperature of 30-40 degrees Celsius for 5-10 minutes.

5. The method according to claim 1, characterized in that, The pH of the HAc-NaAc buffer solution is 4.8-5.2; the concentration of the TMB solution is 5-10.0 mmol / L; and the concentration of the MnO2@MIP nanozyme is 40-50 μg / mL.

6. The method according to claim 1, characterized in that, The sample to be tested is a protein sample; The protein sample contains at least one of the following: urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, and Cu. 2+ Ca 2+ Mg 2+ Al 3+ K + Na + ; Optionally, the linear regression equation is A0 = -0.01185C. VC + 1.87528.

7. The method according to claim 1, characterized in that, The MnO2@MIP nanozyme was prepared by the following method: MnO2 nanosheets were obtained based on the mixed reaction of tetramethylammonium hydroxide, hydrogen peroxide and manganese chloride; The MnO2 nanosheets, N-n-octyl-D-glucosamine, and vitamin C were mixed and subjected to a first reaction at 20-30 °C. Then, the mixture was mixed with ethylene glycol dimethacrylate and subjected to a second reaction at 65-75 °C. The vitamin C was removed by washing to obtain MnO2@MIP nanozyme.

8. A method for detecting vitamin C in proteins based on nanozymes with oxidase activity, characterized in that, include: MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested were mixed, and vitamin C of different concentration gradients was added to obtain mixed solutions. The mixed solution was reacted at a temperature of 30-40 degrees Celsius for 5-10 minutes, and the absorbance of the reaction product at a wavelength of 640-660 nanometers was measured. The vitamin C content in the sample to be tested was determined based on the absorbance values ​​of different mixed solutions. The MnO2@MIP nanozyme was synthesized by the following method: MnO2 nanosheets, N-n-octyl-D-glucosamine, and vitamin C were mixed and subjected to a first reaction at 20-30 °C. Then, the mixture was mixed with ethylene glycol dimethacrylate and subjected to a second reaction at 65-75 °C. The vitamin C was removed by washing to obtain the MnO2@MIP nanozyme.

9. A method for detecting vitamin C in proteins based on nanozymes with oxidase activity, characterized in that, include: Prepare a first mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested, and a second mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and vitamin C at different concentration gradients; The first mixture and the second mixture were reacted at a temperature of 30-40 degrees Celsius for 5-10 minutes, and the absorbance of the reaction products at a wavelength of 640-660 nanometers was measured, corresponding to absorbance values ​​A1 and A0, respectively. A linear regression equation is obtained based on the absorbance value A0 and vitamin C at different concentration gradients. Based on the linear regression equation and the absorbance value A1, the vitamin C content in the sample to be tested is determined. The MnO2@MIP nanozyme was synthesized by the following method: The MnO2 nanosheets, N-n-octyl-D-glucosamine, and vitamin C were mixed and subjected to a first reaction, followed by a second reaction with ethylene glycol dimethacrylate. The vitamin C was then washed away to obtain the MnO2@MIP nanozyme.

10. A MnO2@MIP nanozyme for colorimetric detection of vitamin C, characterized in that, It is prepared by the following method: MnO2 nanosheets, N-n-octyl-D-glucosamine, and vitamin C were mixed and subjected to a first reaction at 20-30 °C. Then, the mixture was mixed with ethylene glycol dimethacrylate and subjected to a second reaction at 65-75 °C. The vitamin C was removed by washing to obtain the MnO2@MIP nanozyme.

11. A vitamin C test kit, characterized in that, include: MnO2@MIP nanozyme, HAc-NaAc buffer and TMB solution, wherein the MnO2@MIP nanozyme is the MnO2@MIP nanozyme according to claim 10.

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