Method for detecting vitamin C based on oxidase active nano enzyme

Abstract

The invention provides a method for detecting vitamin C based on oxidase active nano enzyme. The method comprises the following steps: preparing a first mixture formed by mixing MnO2 (at) MIP nano-enzyme, a TMB solution, a HAc-NaAc buffer solution and a sample to be detected, and a second mixture formed by mixing MnO2 (at) MIP nano-enzyme, a TMB solution, a HAc-NaAc buffer solution and vitamin C with different concentration gradients; reacting the first mixture and the second mixture under the same predetermined reaction condition, and measuring the absorbance of a product after reaction under a predetermined wavelength to respectively correspond to absorbance values A1 and A0; obtaining a linear regression equation based on the A0 and the vitamin C with different concentration gradients, and determining the content of the vitamin C in the sample to be detected based on the linear regression equation and the A1. The method provided by the invention is rapid in detection and extremely high in sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of food detection, and in particular relates to a method for detecting vitamin C based on oxidase activity nanozyme. Background Art

[0002] Vitamin C (VC) is an important water-soluble vitamin that is widely present in fresh fruits and vegetables and is essential for human health. It is involved in a variety of biochemical and physiological processes, including the absorption of metal ions, the synthesis of collagen, and the maintenance of normal secretion of blood vessels and adrenal glands. In addition, the powerful free radical scavenging ability of VC helps maintain the redox balance in the body. Inadequate intake of vitamin C may lead to serious health problems such as scurvy, cancer, Alzheimer's disease, Parkinson's disease, and cardiovascular disease. Therefore, the content of vitamin C in fruits and vegetables is an important indicator for evaluating their quality. In order to accurately evaluate the vitamin C content in fruits and vegetables, a variety of sensitive determination methods have been developed, including chromatography, titration, electrochemistry, capillary electrophoresis, enzymatic analysis, fluorescence analysis, and ultraviolet light. These methods have high reproducibility and selectivity and can achieve the analysis of trace amounts of vitamin C. Among them, spectroscopic methods have attracted increasing attention due to their low cost and simple operation. Colorimetric sensors based on spectroscopy have become important colorimetric tools due to their ease of operation and visual detection, providing convenience for the evaluation of vitamin C content.

[0003] Colorimetric analytical methods that rely on enzymes are widely used for the detection of a variety of targets due to their high selectivity and ease of operation. However, the use of natural enzymes is limited by a variety of factors, including environmental conditions and the molecular structure of the enzyme, which may lead to enzyme inactivation. In addition, the preparation and purification processes of natural enzymes are usually complicated and costly, which severely limits their feasibility in large-scale applications. Therefore, mimetic enzymes with mimetic activity have attracted widespread attention as an alternative. In recent years, studies have found that a variety of nanomaterials (such as noble metal nanoparticles, metal oxides, transition metal carbides, and metal-organic frameworks, etc.) have been found to have 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] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. The present invention provides an efficient and accurate vitamin C detection method based on oxidase drive. The present invention provides a MnO 2 @MIP nanozyme, which can generate reactive oxygen species (ROS) and catalyze the conversion of colorless 3,3',5,5'-tetramethylbenzidine (TMB) into a blue oxidized product (oxTMB). When vitamin C (VC) is present, VC can fill MnO2 The specific imprinted cavity of @MIP allows TMB to interact with the catalytic center MnO 2 The contact is blocked, inhibiting the oxidation of TMB; at the same time, VC, as an antioxidant, can remove ROS, thus fading the blue oxTMB. In summary, a method for detecting vitamin C based on oxidase activity nanozyme adsorption was developed through colorimetric reaction, which can be used to accurately detect the content of vitamin C.

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

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

[0007] Preparation of MnO 2 The first mixture of MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested, and MnO 2 @The second mixture of MIP nanozyme, TMB solution, HAc-NaAc buffer and vitamin C with different concentration gradients;

[0008] The first mixture and the second mixture are reacted under the same predetermined reaction conditions, and the absorbance of the reaction product at a predetermined wavelength is measured, corresponding to absorbance values ​​A and B, respectively. 1 and absorbance value A 0 ;

[0009] Based on the absorbance value A 0 and different concentration gradients of vitamin C to obtain a linear regression equation, based on the linear regression equation and the absorbance value A 1 , determine the content of vitamin C in the sample to be tested;

[0010] The MnO 2 @MIP nanozyme was synthesized by the following method:

[0011] MnO 2 The nanosheets, N-n-octyl-D-glucosamine and vitamin C are mixed for a first reaction, and then mixed with ethylene glycol dimethacrylate for a second reaction, and the vitamin C is washed to remove the MnO 2 @MIP nanozyme.

[0012] According to an embodiment of the present invention, the method for detecting vitamin C based on oxidase activity nanozyme 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 condition is to react 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 is 4.8-5.2; the concentration of the TMB solution is 5-10.0 mmol / L; and the MnO 2 @The concentration of MIP nanozyme is 40-50 μg / mL; the total amount 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 MnO 2 @MIP nanozyme was prepared by the following method:

[0018] Based on the mixed reaction of tetramethylammonium hydroxide, hydrogen peroxide and manganese chloride, MnO 2 Nanosheets;

[0019] MnO 2 The nanosheets, N-octyl-D-glucosamine and vitamin C are mixed and reacted at a temperature of 20-30°C, and then mixed with ethylene glycol dimethacrylate and reacted at a temperature of 65-75°C. The vitamin C is washed and removed to obtain MnO 2 @MIP nanozyme.

[0020] According to an embodiment of the present invention, the MnO 2 @MIP nanozyme was prepared by the following method:

[0021] S1: Tetramethylammonium hydroxide solution, H 2 O 2 The solution was mixed with deionized water and then MnCl 2 ·4H 2 O solution, react, wash to obtain block manganese dioxide, then add the block manganese dioxide into deionized water to dissolve the obtained solution, and freeze-dry to obtain MnO 2 Nanosheets;

[0022] S2: The prepared MnO 2 Nanosheets, N-octyl-D-glucosamine and vitamin C are added to dichloromethane, stirred and reacted at 20-30°C for 6-8h, and then ethylene glycol dimethacrylate is added, stirred and reacted at 65-75°C for 11-13 hours to obtain an intermediate product, which is washed and dried to obtain the MnO 2 @MIP nanozyme.

[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 protein. The protein sample contains at least one of the following: urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, Cu 2+ , Ca 2+ Mg 2+ 、Al 3+ , K + 、Na + The presented method is not interfered by these substances.

[0024] The second aspect of the present invention provides a method for detecting vitamin C in protein based on oxidase activity nanozyme, comprising:

[0025] Preparation of MnO 2 The first mixture of MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested, and MnO 2 @The second mixture of MIP nanozyme, TMB solution, HAc-NaAc buffer and vitamin C with different concentration gradients;

[0026] The first mixture and the second mixture are 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 is measured, corresponding to the absorbance values ​​A and B, respectively. 1 and absorbance value A 0 ;

[0027] Based on the absorbance value A 0 and different concentration gradients of vitamin C to obtain a linear regression equation, based on the linear regression equation and the absorbance value A 1 , determine the content of vitamin C in the sample to be tested;

[0028] The MnO 2 @MIP nanozyme was synthesized by the following method:

[0029] MnO 2 The nanosheets, N-octyl-D-glucosamine and vitamin C are mixed for a first reaction, and then mixed with ethylene glycol dimethacrylate for a second reaction, and the vitamin C is washed to remove the vitamin C, so as to obtain MnO 2 @MIP nanozyme.

[0030] The third aspect of the present invention provides a method for detecting vitamin C in protein based on oxidase activity nanozyme, comprising:

[0031] MnO 2@MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested are mixed, and vitamin C with different concentration gradients is added to obtain a mixed solution;

[0032] The mixed solution is 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 is measured;

[0033] Determine the content of vitamin C in the sample to be tested based on the absorbance values ​​of different mixed solutions;

[0034] The MnO 2 @MIP nanozyme was synthesized by the following method:

[0035] Tetramethylammonium hydroxide solution, hydrogen peroxide solution and manganese chloride solution are mixed and reacted to obtain MnO 2 Nanosheets;

[0036] The MnO 2 Nanosheets, N-octyl-D-glucosamine and vitamin C were reacted, and then reacted with ethylene glycol dimethacrylate to obtain MnO 2 @MIP nanozyme.

[0037] A fourth aspect of the present invention provides a MnO for colorimetric detection of vitamin C. 2 @MIP nanozyme is prepared by the following method:

[0038] MnO 2 The nanosheets, N-octyl-D-glucosamine and vitamin C are mixed for a first reaction, and then mixed with ethylene glycol dimethacrylate for a second reaction, and the vitamin C is washed to remove the vitamin C, so as to obtain MnO 2 @MIP nanozyme.

[0039] A fifth aspect of the present invention provides a vitamin C detection kit, comprising: MnO 2 @MIP nanozyme, HAc-NaAc buffer, TMB solution. The vitamin C detection kit may also include vitamin C standard as needed.

[0040] The beneficial effects achieved by the present invention are at least:

[0041] (1) The method of the present invention is used to detect vitamin C, and the detection time is shorter. The color change of the detection 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 require the addition of H 2 O 2 , effectively avoiding the direct addition of unstable H 2O 2 Operation caused false positive output.

[0043] (3) MnO used in the present invention 2 @MIP nanozyme exhibits outstanding performance in selectivity and anti-interference. Its three-dimensional cavity with high adaptability to VC molecules excludes representative substrates in proteins and VC structural analogs such as urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, Cu 2+ , Ca 2+ Mg 2+ 、Al 3+ , K + 、Na + It is not affected by interference, and has good selectivity and anti-interference.

[0044] (4) The detection method adopted by the present invention has extremely high sensitivity, and the detection limit LOD is as low as 0.253 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 According to the embodiment of the present invention, MnO 2 @TEM image of MIP.

[0046] Figure 2 According to the embodiment of the present invention, MnO 2 @MIP characterization diagram. Figure A is MnO 2 @Infrared spectrum of MIP, Figure B is MnO 2 @MIP fluid dynamics, Figure C is MnO 2 @Zeta potential of MIP, Figure D is MnO 2 @MIP’s full XPS spectrum.

[0047] Figure 3 The MnO provided by the embodiment of the present invention 2 @MIP's XPS elemental high-resolution spectrum, including C, N, O, and Mn.

[0048] Figure 4 The MnO provided by the embodiment of the present invention 2 @Optimization of conditions for detecting VC using the MIP+TMB system, including pH value and temperature.

[0049] Figure 5 The MnO provided by the embodiment of the present invention 2 @The feasibility of MIP+TMB system for detecting VC was verified in different groups; (1) VC, (2) TMB, (3) MnO 2 @MIP,(4)MnO 2@MIP+TMB,(5)MnO 2 UV absorption graph of @MIP+TMB+VC and the corresponding solution color illustration.

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

[0051] Figure 7 The MnO provided by the embodiment of the present invention 2 @MIP+TMB detection system selectivity and anti-interference test. Figures A 1-15 are blank, VC, urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, Cu 2+ , Ca 2+ Mg 2+ 、Al 3+ , K + 、Na + ; 1-14 in Figure B are VC+ blank, urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, Cu 2+ , Ca 2+ Mg 2+ 、Al 3+ , K + 、Na + . DETAILED DESCRIPTION

[0052] 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 are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0053] The present invention provides a method for detecting vitamin C based on oxidase activity nanozyme to detect the vitamin C content in protein, which has important biological and medical significance. First, vitamin C (ascorbic acid) plays a variety of key roles in the human body, including promoting the synthesis of collagen, enhancing immunity, anti-oxidation, and participating in the activity regulation of multiple enzymes. Vitamin C deficiency can lead to serious health problems such as scurvy, decreased immunity, and poor wound healing. Detecting the vitamin C content in protein can help evaluate the nutritional value of food or biological samples. Since vitamin C cannot be synthesized in the human body and must be ingested through diet, its content is one of the important indicators for evaluating the nutritional value of food. In addition, the detection of vitamin C is also crucial to ensure the quality of food and medicine. In addition, vitamin C, as a cofactor of multiple hydroxylases, is crucial to the synthesis of collagen, and collagen is the most abundant protein in the human body and plays a key role in the structural integrity of systemic tissues. Therefore, detecting the vitamin C content in protein helps to understand its functional state in vivo, especially when studying diseases related to collagen. Detecting the vitamin C content in protein not only helps to assess nutritional intake and food quality, but also helps to understand the physiological functions of vitamin C in organisms and the possible health effects of its deficiency or excess.

[0054] The present invention provides a MnO 2 @MIP nanozyme, which is synthesized by the following method:

[0055] MnO 2 The nanosheets, N-octyl-D-glucosamine and vitamin C are mixed for a first reaction, and then mixed with ethylene glycol dimethacrylate for a second reaction, and the vitamin C is washed to remove the vitamin C, so as to obtain MnO 2 @MIP nanozyme.

[0056] The synthesis method of the provided nanozyme is to add vitamin C as a synthesis template, wrap it around the nanozyme, and then remove the vitamin C by elution, thereby leaving a specific cavity of vitamin C, which is specific to vitamin C, so as to achieve the purpose of detecting and adsorbing vitamin C. Compared with other nanozymes, the detection time is short, the selectivity is better, and multiple interferences are not affected.

[0057] According to a specific embodiment, the provided method for detecting vitamin C based on oxidase activity nanozyme comprises the following steps:

[0058] Step 1: Determine the absorbance of the test solution of vitamin C standard with different concentration gradients at 650nm:

[0059] At room temperature, MnO 2@MIP nanozyme, TMB and different concentrations of vitamin C were added to the HAc-NaAc buffer for reaction. The mixture was stirred thoroughly and the reaction lasted for 5.0 min before being transferred to a cuvette. The absorbance at 650 nm was then measured using a UV spectrophotometer and recorded as A 0 ;

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

[0061] The UV regression equation A was constructed using the absorbance values ​​measured by the vitamin C standards with different concentration gradients and the concentration of the standards. 0 =XC VC +y, where C VC is the concentration of the standard;

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

[0063] In step 1, the pH value of the HAc-NaAc buffer solution is 4.8-5.2; the concentration of the TMB solution is 10.0 mmol / L; and the MnO 2 @The concentration of MIP nanozyme is 50.0 μg / mL; the total amount of the mixed solution is 200.0 μL; the reaction temperature is 35.0°C; and the reaction time is 5.0 min.

[0064] According to a specific embodiment, the linear regression equation is A 0 =-0.01185C VC +1.87528;

[0065] According to a specific embodiment, the MnO 2 The preparation method of @MIP nanozyme includes the following steps:

[0066] S1: Tetramethylammonium hydroxide solution (TMAH, 25 wt%) and H 2 O 2 The solution (3 wt%) was thoroughly mixed with deionized water, and then MnCl 2 ·4H 2 O solution. Stir overnight in the open air. Then, wash with ultrapure water and methanol three times, collect bulk manganese dioxide after centrifugation, purification and drying. Then, the bulk MnO 2 Add deionized water, ultrasonicate for 1-3h, and freeze-dry the resulting solution to obtain MnO 2 Nanosheets.

[0067] S2: Take the prepared MnO 2Nanosheets, N-n-octyl-D-glucosamine and vitamin C are added to dichloromethane (DCM), stirred at 20-30°C for 6-8h, and then ethylene glycol dimethacrylate is added, stirred at 65-75°C for 11-13h to obtain an intermediate product, and the vitamin C in the intermediate product is removed by washing, filtered, and dried at 65-75°C to obtain a molecular imprinting polymer MnO for adsorbing vitamin C. 2 @MIP.

[0068] The technical scheme of the present invention is described below by specific embodiments. It should be noted that the following embodiments are provided to better understand the present invention, but are not intended to limit the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified.

[0069] Related reagents used in the embodiments of the present invention:

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

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

[0072] Example 1

[0073] Example 1: Synthetic MnO was prepared by the following method 2 @MIP nanozymes, including:

[0074] (1) Tetramethylammonium hydroxide solution (TMAH, 25 wt%) and H 2 O 2 The solution (3 wt%) was thoroughly mixed with deionized water, and then MnCl 2 ·4H 2 O solution. Stir overnight in the open air. Then, wash with ultrapure water and methanol three times, collect bulk manganese dioxide after centrifugation, purification and drying. Then, the bulk MnO 2 Add deionized water, ultrasonicate for 2 h, and freeze-dry the resulting solution to obtain MnO 2 Nanosheets.

[0075] (2) Take the prepared MnO 2 Nanosheets, N-octyl-D-glucosamine and vitamin C were added to dichloromethane (DCM) and stirred at 25 °C for 1 h. Ethylene glycol dimethacrylate was then added and stirred at 70 °C for 12 h. The obtained product was washed with methanol for 2 hours to remove the vitamin C in the intermediate product. After filtering, it was dried at 70 °C to obtain MnO 2 @MIP nanozyme.

[0076] Example 2

[0077] Example 2 Using the MnO prepared in Example 1 2 @MIP nanozyme optimized the conditions for detecting vitamin C, especially the experimental parameters such as pH value and temperature in the detection system, including:

[0078] The experiment first explored the performance of the system in the pH range of 3.5-5.5, and HAc-NaAc buffer was selected as the reaction buffer. Figure 4 As shown in A, the results show that when the pH value is in the range of 3.5 to 5.0, the absorbance value of the reaction system at 650nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 5.0, the absorbance value decreases as it increases, so 5.0 is selected as the optimal pH value of the system.

[0079] Next, the performance of the system in the temperature range of 25.0-45.0℃ was investigated, and the results are as follows: Figure 4 As shown in B. The experimental results show that when the temperature is in the range of 25.0℃ to 35.0℃, the absorbance value of the reaction system at 650nm increases with the increase of the reaction temperature. When the temperature exceeds 35.0℃ and increases to 45.0℃, the absorbance value decreases as it increases, so 35.0℃ is selected as the optimal temperature of the system.

[0080] Example 3

[0081] Example 3: For the use of MnO 2 The feasibility of @MIP nanoenzyme to detect vitamin C was verified, including:

[0082] Use TMB as the colorimetric agent and HAc-NaAc as the buffer. Take the following five groups of reactions. The group numbers are:

[0083] (1) VC, (2) TMB, (3) MnO 2 @MIP, (4)MnO 2 @MIP+TMB, (5)MnO 2@MIP+TMB+VC. After sufficient reaction, transfer to a cuvette and measure the absorbance at 650nm with a UV spectrophotometer to compare the values.

[0084] Results Figure 5 , indicating that vitamin C can inhibit the oxidase activity and cause a significant change in absorbance. The experiment shows the feasibility of the detection system proposed by the present invention.

[0085] In the above verification step, MnO was added 2 @The concentration of MIP nanozyme is 50.0 mg / mL; the concentration of TMB is 10.0 mmol / L; the concentration of VC is 120 mM; the pH of the added HAc-NaAc buffer is 4.8-5.2; the volume of the total mixed solution is 200.0 mL; the reaction temperature is 35.0°C; and the time for uniform mixing and sufficient reaction is 5.0 min.

[0086] Example 4

[0087] Example 4 Using MnO 2 @MIP nanoenzyme detects different concentrations of vitamin C standards, 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 mixed with 50.0 μg / mL of MnO 2 @MIP nanozyme and 10.0 mmol / L TMB solution were fully reacted in HAc-NaAc buffer (pH = 4.8-5.2) at 35.0°C for 5.0 min.

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

[0090] The results are as follows Figure 6 As shown, the concentration of VC and absorbance A are linear in the range of 0-120.0 mM, and the absorbance regression equation is A = -0.01185C VC +1.87528, the correlation coefficient is 0.99322, and the detection limit is 0.253 μM. In addition, as the concentration of VC increases, the solution presents a color gradient (dark blue to light blue), and the change in the color of the reagent can be observed by the naked eye, thus realizing the detection of VC concentration.

[0091] Example 5

[0092] Example 5 tested the selectivity and anti-interference of the method provided by the present invention for the detection of vitamin C, using some representative contents as concept verification, including 1-15 corresponding to blank, VC, urea, glucose, citric acid, serine, glutamic acid, arginine, glycine, Cu 2+ , Ca 2+ Mg 2+ 、Al 3+ , K + 、Na + .include:

[0093] At room temperature, MnO 2 @MIP nanozyme and TMB were reacted with the above substances in HAc-NaAc buffer respectively; after mixing evenly, reacted for 5.0 min and then transferred to a cuvette. The absorbance value A at 650 nm was measured respectively. The results are shown in Figure 7 As shown in A, only the absorbance value A of VC decreased significantly, indicating that the detection system has good selectivity for VC. At the same time, we explored whether the coexistence of VC and these substances would affect the reaction system. The results are shown in Figure 7 As shown in B, there is no obvious change in the absorbance value at 650nm of each coexistence system, indicating that the constructed detection system has good anti-interference ability.

[0094] The added MnO 2 @The concentration of MIP nanozyme is 50.0 μg / mL; the concentration of TMB is 10.0 mmol / L; the concentration of the selectivity verification substance is 120.0 mM; the pH of the added HAc-NaAc buffer is 4.8-5.2; the total mixed solution is 200.0 mL; the reaction temperature is 35.0 °C; and the time for uniform mixing and sufficient reaction is 5.0 min.

[0095] Example 6

[0096] Example 6: Vitamin C in actual samples was detected using the method provided by the present invention.

[0097] Taking soybean protein (purchased randomly from a large supermarket in Hefei, China, without purification) as a representative, 1g of soybean protein was added to 10.0mL of deionized water and adjusted to pH = 5.0. Then 10.0, 60.0, and 120.0mM VC were added respectively. At room temperature, MnO 2 @MIP and TMB were reacted with the above mixed solution in HAc-NaAc buffer respectively; after being mixed evenly, the mixture was reacted for 5.0 min and then transferred to a cuvette. The absorbance value A at 650 nm was measured respectively. The obtained absorbance value A was substituted into the corresponding linear equation, and the VC results measured in the 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. 1g of pea protein was added to 10.0mL of deionized water and adjusted to pH = 5.0. Then 10.0, 60.0, and 120.0mM VC were added respectively. At room temperature, MnO 2 @MIP and TMB were reacted with the above mixed solution in HAc-NaAc buffer respectively; after being mixed evenly, the mixture was reacted for 5.0 min and then transferred to a cuvette. The absorbance value A at 650 nm was measured respectively. The obtained absorbance value A was substituted into the corresponding linear equation, and the VC results measured in the 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. 1g chickpea protein was added to 10.0mL deionized water and adjusted to pH = 5.0. Then 10.0, 60.0, and 120.0mM VC were added respectively. At room temperature, MnO 2 @MIP and TMB were reacted with the above mixed solution in HAc-NaAc buffer respectively; after being mixed evenly, the mixture was reacted for 5.0 min and then transferred to a cuvette. The absorbance value A at 650 nm was measured respectively. The obtained absorbance value A was substituted into the corresponding linear equation, and the VC results measured in the colorimetric detection mode are shown in Table 1.

[0100] Table 1 shows MnO 2 @Detection of VC in actual samples by MIP+TMB system

[0101]

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

[0103] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific implementation" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0104] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for detecting vitamin C based on oxidase activity nanozyme, characterized in that: include: Prepare a first mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and a sample to be tested, and a second mixture of MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and vitamin C with 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, which correspond to absorbance values ​​A1 and A0, respectively; Obtaining a linear regression equation based on the absorbance value A0 and vitamin C of different concentration gradients, and determining the content of vitamin C in the sample to be tested based on the linear regression equation and the absorbance value A1; The MnO2@MIP nanozyme is synthesized by the following method: The MnO2 nanosheets, N-n-octyl-D-glucosamine and vitamin C are mixed for a first reaction, and then mixed with ethylene glycol dimethacrylate for a second reaction, and the vitamin C is washed to remove the MnO2@MIP nanozyme.

2. The method according to claim 1, characterized in that: The predetermined wavelength is 640-660 nanometers, preferably 650 nanometers.

3. The method according to claim 1, characterized in that The predetermined reaction condition is to react at a temperature of 30 to 40 degrees Celsius for 5 to 10 minutes.

4. The method according to claim 1, characterized in that 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; and the concentration of the MnO2@MIP nanozyme is 40-50 μg / mL.

5. 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, Cu 2+ , Ca 2+ Mg 2+ 、Al 3+ , K + 、Na + ; Optionally, the linear regression equation is A0=-0.01185C VC +1.87528.

6. The method according to claim 1, characterized in that The MnO2@MIP nanozyme is 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-octyl-D-glucosamine and vitamin C are mixed and subjected to a first reaction at a temperature of 20-30°C, and then mixed with ethylene glycol dimethacrylate and subjected to a second reaction at 65-75°C, and the vitamin C is washed away to obtain the MnO2@MIP nanozyme.

7. A method for detecting vitamin C in protein based on oxidase activity nanozyme, characterized in that: include: The MnO2@MIP nanozyme, TMB solution, HAc-NaAc buffer and the sample to be tested were mixed, and vitamin C with different concentration gradients was added to obtain a mixed solution; The mixed solution is 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 is measured; Determine the content of vitamin C in the sample to be tested based on the absorbance values ​​of different mixed solutions; The MnO2@MIP nanozyme is synthesized by the following method: The MnO2 nanosheets, N-octyl-D-glucosamine and vitamin C are mixed and subjected to a first reaction at a temperature of 20-30°C, and then mixed with ethylene glycol dimethacrylate and subjected to a second reaction at 65-75°C, and the vitamin C is washed to remove the vitamin C, so as to obtain the MnO2@MIP nanozyme.

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

9. A MnO2@MIP nanozyme for colorimetric detection of vitamin C, characterized in that: Prepared by the following method: The MnO2 nanosheets, N-octyl-D-glucosamine and vitamin C are mixed and subjected to a first reaction at a temperature of 20-30°C, and then mixed with ethylene glycol dimethacrylate and subjected to a second reaction at 65-75°C, and the vitamin C is washed to remove the vitamin C, so as to obtain the MnO2@MIP nanozyme.

10. A vitamin C detection kit, characterized in that: include: MnO2@MIP nanozyme, HAc-NaAc buffer and TMB solution.

Images