Application of Iron-Melanin Peroxidase Mimic Enzyme in Detecting Total Antioxidant Capacity

Abstract

The present invention provides a method for applying iron-melanin as a peroxidase mimetic enzyme in the detection of total antioxidant capacity, belonging to the technical field of detection and analysis. The iron-melanin prepared in the present invention can catalyze the oxidation of hydrogen peroxide to colorimetric substrates. After adding antioxidants, the oxidized colorimetric substrates are reduced. By measuring the absorbance values before and after the reduction reaction and analyzing the relationship between the absorbance values and the antioxidant concentration, a linear equation between the two can be established to detect the total antioxidant capacity of the sample. The iron-melanin prepared in the present invention as a peroxidase mimetic enzyme has more excellent catalytic performance compared with HRP, and also has excellent stability and reusability. According to the method provided by the present invention for detecting the total antioxidant capacity, the lowest detection limit is 12.61 μmol / L.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis, and specifically relates to the application of iron-melanin as a peroxidase mimetic enzyme in the detection of total antioxidant capacity. Background Art

[0002] Horseradish peroxidase (HRP) has obvious advantages compared with similar peroxidase enzymes. Due to its high sensitivity, fast analysis speed, and low background interference, it has been widely used in biosensors for the detection of H2O2, glucose, acetylcholine, and ascorbic acid. However, during the detection process, the exposed active center of HRP is easily affected by temperature, pH value, H2O2, and adsorption of reaction by-products, bringing great uncertainty to the detection results. Although many immobilization strategies have been developed to protect the enzyme activity and increase its stability, due to the relatively low enzyme loading during the immobilization process and serious loss of enzyme activity, the catalytic effect of the material is insufficient.

[0003] Inspired by natural enzymes, many nanomaterials with similar peroxidase catalytic activity have been developed as mimetic enzymes to construct new biosensors. However, most of the previous mimetic enzymes based on nanomaterials have problems such as high cost or complex preparation processes. For example, Chinese Patent Application CN113617395A discloses a nanozyme for detecting the antioxidant activity of food and its preparation method. Potassium hexacyanoferrate and citric acid are dissolved in water, and then sodium tetrachloroaurate (III) and copper sulfate are added in sequence, and a gold-doped nanozyme Au@Cu-HCF is obtained by reaction. This nanozyme can be used to detect both hydrogen atom transfer type antioxidant substances and single electron transfer type antioxidant substances. However, although noble metal-based nanozymes have high peroxidase activity, their high price and scarcity limit their wide application.

[0004] Therefore, the safety of mimetic enzymes and the simplicity of synthesis methods still need to be further improved; there is an urgent need to develop artificial peroxidase mimetic enzymes with high activity, simple preparation methods, low cost, and good stability to avoid the disadvantages of natural enzymes. Summary of the Invention

[0005] To solve the above problems, in the present invention, iron-melanin is used as a peroxidase mimetic enzyme to catalyze the oxidation of hydrogen peroxide to colorimetric substrates, and the mechanism that antioxidants can reduce the oxidized colorimetric substrates is utilized to achieve the rapid detection of total antioxidant capacity.

[0006] To achieve the above purpose, the method for using iron-melanin as a peroxidase mimetic enzyme to detect total antioxidant capacity in the present invention includes the following steps:

[0007] S1. Add hydrogen peroxide, a chromogenic substrate, and iron-melanin to a sodium acetate - acetic acid buffer solution, and stir evenly to obtain a mixed solution A. The concentration of hydrogen peroxide in the mixed solution A is 1.0 mmol / L, the concentration of the chromogenic substrate is 1.0 mmol / L, and 0 < the concentration of iron-melanin ≤ 50 μg / mL;

[0008] S2. Take N portions of the mixed solution A, add aqueous solutions of antioxidants with different concentrations respectively, stir evenly, and incubate statically to obtain N test solutions B with different antioxidant concentrations; the concentration of the antioxidant in the test solution B is 0 - 0.3 mmol / L, and the test solution B with an antioxidant concentration of 0 is used as the control group;

[0009] S3. Measure the ultraviolet-visible absorption spectra of the N test solutions B in step S2 in the range of 350 - 800 nm, and record the absorbance values of the control group and the other test solutions B at 652 nm as Y0, Y1, Y2... Y N-1 ; Calculate the differences between Y1, Y2... Y N-1 and Y0 respectively, conduct data analysis on the absorbance differences and antioxidant concentrations, establish a model relationship between the antioxidant concentration x and the absorbance difference Y, and obtain the standard curve for detecting the antioxidant as Y = 0.00189x + 0.03456;

[0010] S4. Prepare a sample solution with an unknown antioxidant concentration according to the methods in steps S1 and S2, measure the absorbance value of the sample solution at 652 nm, substitute the difference between the obtained absorbance value and the absorbance value Y0 of the control group into the standard curve in step S3, and calculate the concentration of the antioxidant.

[0011] In the present invention, iron-melanin exerts peroxidase-like activity under the acidic conditions provided by acetate buffer, catalyzing the decomposition of hydrogen peroxide to generate ·OH to oxidize the chromogenic substrate. Then, different concentrations of antioxidants are used to reduce the oxidized chromogenic substrate, and the absorbance value of the reduced system at 652 nm is measured. The oxidized chromogenic substrate solution without adding antioxidants is used as a control group, and its absorbance value is measured. The difference between the absorbance value of the reduced system and the absorbance value of the control group is calculated, and the relationship between the difference and the antioxidant concentration is analyzed. A linear equation between the two is established to obtain a standard curve for detecting the antioxidant concentration. Prepare the oxidized chromogenic substrate solution according to the method of step S1. Add the test sample with a concentration within the detection range to the oxidized chromogenic substrate solution according to the method of step S2, measure the absorbance value of the mixed system at 652 nm, then calculate the difference between this absorbance value and the absorbance value of the above control group, and substitute the difference into the standard curve to calculate the concentration of the antioxidant in the test sample. In the test sample with ascorbic acid as the main antioxidant component, the concentration of ascorbic acid is detected, and the total antioxidant capacity of the sample is obtained by equating the antioxidant capacity of ascorbic acid to the total antioxidant capacity of the sample. The method for detecting the total antioxidant capacity in the present invention is applicable to detecting the total antioxidant capacity of various test samples such as drugs, health products, foods, and cosmetics with ascorbic acid as the main antioxidant component.

[0012] Preferably, the pH of the sodium acetate-acetic acid buffer is 2-6.

[0013] More preferably, the pH of the sodium acetate-acetic acid buffer is 2-5.

[0014] Even more preferably, the pH of the sodium acetate-acetic acid buffer is 2-4.

[0015] Even further preferably, the pH of the sodium acetate-acetic acid buffer is 3.

[0016] Preferably, the temperature for preparing the test solution B in step S2 is 35-70 °C.

[0017] More preferably, the temperature for preparing the test solution B in step S2 is 40-65 °C.

[0018] Even more preferably, the temperature for preparing the test solution B in step S2 is 50 °C.

[0019] Preferably, the static incubation time in step S2 is 5-25 min.

[0020] More preferably, the static incubation time in step S2 is 15-25 min.

[0021] Preferably, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine.

[0022] Preferably, the antioxidant is ascorbic acid.

[0023] Preferably, the method for preparing iron-melanin comprises the following steps:

[0024] P1. Add cuttlefish ink into deionized water, stir until the cuttlefish ink is evenly suspended, wash the obtained suspension with deionized water and centrifuge 5-7 times, and then freeze-dry to obtain melanin nanoparticles;

[0025] P2. Resuspend the melanin nanoparticles in deionized water, add an aqueous solution containing Fe 3+ to obtain a mixed solution C, adjust the pH of the mixed solution C to 3 with a pH regulator, and stir and react for 6 h; the concentration of melanin nanoparticles in the mixed solution C is 12.5 mg / mL; the concentration of Fe 3+ in the mixed solution C is 9.6 mmol / mL;

[0026] P3. Centrifuge and wash the aggregate solution obtained after the reaction in step P2 with deionized water 5-7 times to obtain an iron-melanin solution, and then freeze-dry to obtain iron-melanin powder.

[0027] More preferably, the concentration of cuttlefish ink in the suspension in step P1 is 0.25 g / mL.

[0028] More preferably, the speed of each centrifugation in step P1 is 10000 r / min, and the time of each centrifugation is 10 min.

[0029] More preferably, the aqueous solution containing Fe 3+ in step P2 is an FeCl3 aqueous solution.

[0030] More preferably, the pH regulator in step P2 is an aqueous sodium hydroxide solution.

[0031] Even more preferably, the concentration of the aqueous sodium hydroxide solution is 0.1 mol / L.

[0032] More preferably, the speed of each centrifugation in step P3 is 10000 r / min, and the time of each centrifugation is 5 min.

[0033] More preferably, the conditions for freeze-drying in steps P1 and P3 are drying at -60°C for 12 h.

[0034] Compared with the prior art, the beneficial effects of the present invention are:

[0035] (1) When iron-melanin is used as a peroxidase mimic enzyme in the present invention, at a concentration of only 10 μg / mL, the Michaelis constant Km of the enzyme kinetics is 0.367, and the maximum reaction rate Vmax can reach 10.1×10 -8 m / s, and its catalytic activity is much greater than that of natural horseradish peroxidase; moreover, the stability and reusability of the iron-melanin peroxidase mimic enzyme in the present invention are very excellent. After being reused 5 times, the enzyme activity decreases by no more than 10%; when stored for 20 days under laboratory conditions (temperature 20 - 30 °C, humidity 45% - 80%), the enzyme activity decreases by no more than 10%.

[0036] (2) When the iron-melanin peroxidase mimic enzyme in the present invention is used to detect the total antioxidant capacity of a sample with ascorbic acid as the main antioxidant component, the lowest detection limit is 12.61 μmol / L, and the detection range is 5 - 250 μmol / L.

[0037] (3) The synthesis method of the iron-melanin peroxidase mimic enzyme provided in the present invention is simple, the operation is convenient, the synthesis time is fast, the raw material sources are extensive, and the cost is low. Description of the Drawings

[0038] Figure 1 is a schematic diagram of the principle for detecting the total antioxidant capacity in the present invention;

[0039] Figure 2 is a transmission electron microscope image of the iron-melanin prepared in the present invention;

[0040] Figure 3 is a graph of the measurement results of the activity of the iron-melanin peroxidase mimic enzyme at different temperatures;

[0041] Figure 4 is a graph of the measurement results of the activity of the iron-melanin peroxidase mimic enzyme at different pH values;

[0042] Figure 5 is a graph of the measurement results of the activity of the iron-melanin peroxidase mimic enzyme with different concentrations changing with the reaction time;

[0043] Figure 6 is a graph of the Michaelis kinetic constant fitting curve of the iron-melanin peroxidase mimic enzyme at different TMB concentrations;

[0044] Figure 7 is a graph of the reaction rate of the iron-melanin peroxidase mimic enzyme at different TMB concentrations;

[0045] Figure 8 is a graph of the Michaelis kinetic constant fitting curve of the iron-melanin peroxidase mimic enzyme at different hydrogen peroxide concentrations;

[0046] Figure 9Reaction rate diagram of iron-melanin peroxidase mimic enzyme at different hydrogen peroxide concentrations;

[0047] Figure 10 Diagram of the determination results of the stability of iron-melanin peroxidase mimic enzyme;

[0048] Figure 11 Diagram of the determination results of the reusability of iron-melanin peroxidase mimic enzyme;

[0049] Figure 12 Standard curve diagram for the detection of total antioxidant capacity;

[0050] Figure 13 Diagram comparing the results of the detection of the total antioxidant capacity of two vitamin C tablets with the content in the specification by the method of the present invention;

[0051] Figure 14 Diagram of the detection results of the total antioxidant capacity of three beverages, oranges and lemons by the method of the present invention. Detailed implementation mode

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the scope protected by the present invention. Any equivalent transformation or substitution made by those skilled in the art according to the following implementation modes belongs to the scope of protection of the present invention.

[0053] Embodiment

[0054] 1. Preparation of iron-melanin peroxidase mimic enzyme

[0055] The method for preparing iron-melanin in this embodiment includes the following steps:

[0056] P1. Add 25 g of cuttlefish ink into 100 mL of deionized water, stir until the cuttlefish ink is evenly suspended, wash the obtained suspension with deionized water at a centrifugal speed of 10,000 r / min for 10 min, repeat the washing 5 - 7 times, and then freeze-dry at -60 °C for 12 h to obtain dry melanin nanoparticles;

[0057] P2. Suspend 0.5 g of the melanin nanoparticles obtained in step P1 in 35 mL of water, slowly add 5 mL of 0.077 mol / L FeCl3 aqueous solution, stir evenly, add 0.1 mol / L NaOH solution to adjust the pH to 3, and magnetically stir and react for 6 h to obtain a solution of Fe 3+ Modified melanin nanoparticle aggregate solution;

[0058] P3. Wash the aggregate solution in step P2 with deionized water at a centrifugal speed of 10,000 r / min for 5 min, and repeat the washing 5 - 7 times to obtain an iron-melanin solution with peroxidase-like activity. Freeze-dry it at -60 °C for 12 h to obtain iron-melanin (Fe-NMPs) peroxidase mimicking enzyme powder.

[0059] Figure 2 is the transmission electron microscopy image of the iron-melanin solution obtained in step P3. It can be seen from Figure 2 that the prepared iron-melanin is spherical in shape with a particle size of 100 nm.

[0060] 2. Determination of the performance of iron-melanin (Fe-NMPs) peroxidase mimicking enzyme

[0061] (1) Effect of temperature on the activity of iron-melanin peroxidase mimicking enzyme

[0062] Add 20 μL of 0.25 mg / mL iron-melanin solution, 25 μL of 20 mmol / L TMB solution and 25 μL of 20 mmol / L hydrogen peroxide to 430 μL of sodium acetate - acetic acid buffer (pH = 3). The concentration of iron-melanin in the resulting mixed solution is 10 μg / mL; a total of 6 identical solutions are prepared. Place the 6 identical solutions at 20 °C, 30 °C, 40 °C, 50 °C, 60 °C and 70 °C for reaction for 15 minutes. Starting from the 15th minute, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value of each well's reaction system at 652 nm per minute. The measurement results are as Figure 3 shown. It can be seen from Figure 3 that when the temperature is lower than 50 °C, the catalytic activity of iron-melanin increases with the increase of temperature, and reaches the strongest at 50 °C. After that, with the increase of temperature, the catalytic activity of iron-melanin gradually decreases; however, its catalytic activity is still 50% at 70 °C.

[0063] (2) Effect of pH value on the activity of iron-melanin peroxidase mimicking enzyme

[0064] Take 430 μL of sodium acetate - acetic acid buffer with pH values of 2, 3, 4, 5, 6, 7, 8, and 9 in sequence. Add 20 μL of 0.25 mg / mL iron-melanin solution, 25 μL of 20 mmol / L TMB solution and 25 μL of 20 mmol / L hydrogen peroxide to each buffer solution. The concentration of iron-melanin in the resulting mixed solution is 10 μg / mL. Let the 8 solutions stand and incubate for 15 min. Starting from the 15th minute, use an ELISA reader to measure the absorbance value of each well's reaction system at 652 nm per minute. The measurement results are as Figure 4 shown. It can be seen from Figure 4It can be seen that in the range of pH = 2 - 9, iron-melanin has activity as a peroxidase mimic enzyme; in the range of pH = 2 - 3, the activity of the mimic enzyme gradually increases with the increase of pH value; in the range of pH = 3 - 9, the activity of the mimic enzyme gradually decreases with the increase of pH value; in the range of pH = 3 - 6, the activity decreases rapidly, and in the range of pH = 6 - 9, the activity decreases slowly; when the pH of the reaction system is 3, the catalytic activity of iron-melanin is the strongest.

[0065] (3) Effects of iron-melanin concentration and reaction time on the activity of iron-melanin peroxidase mimic enzyme

[0066] Take 6 portions of sodium acetate - acetic acid buffer solution (pH = 3) with a volume of 170 μL each. Add 10 μL of TMB solution with a concentration of 20 mmol / L and 10 μL of hydrogen peroxide solution with a concentration of 20 mmol / L to each buffer solution, and then add 10 μL of iron-melanin solution with different concentrations respectively, so that the final concentrations of iron-melanin are 0, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL in sequence; among them, the buffer solution system without adding iron-melanin is used as a blank control; within 25 min, use an enzyme-labeling instrument to measure the absorbance of the reaction system at 652 nm in each well per minute, and the measurement results are as Figure 5 shown. It can be seen from Figure 5 that regardless of the concentration of iron-melanin in the buffer solution system, its catalytic activity increases with the prolongation of the reaction time; within the same reaction time, the activity of the iron-melanin peroxidase mimic enzyme increases with the increase of its concentration in the buffer solution system.

[0067] (4) Evaluation of the affinity of iron-melanin for the chromogenic substrate

[0068] Add 10 μL of iron-melanin solution with a concentration of 0.2 mg / mL to 10 portions of sodium acetate - acetic acid buffer solution (pH = 3) respectively, and then add TMB solution with a concentration of 20 mmol / L and 10 μL of hydrogen peroxide solution with a concentration of 20 mmol / L with different volumes, and make up the volume to 200 μL with sodium acetate - acetic acid buffer solution (pH = 3), so that the final concentrations of TMB in the 10 buffer solution systems are 0.1 mmol / L, 0.5 mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, and 4.5 mmol / L in sequence; measure the absorbance of the reaction system at 652 nm in each well every 7.5 s; the measurement results are as Figure 6 and Figure 7 shown. Figure 6 is the Michaelis - Menten kinetic constant fitting curve graph of iron-melanin at different TMB concentrations.Figure 7 Figure showing the reaction rate of iron-melanin at different TMB concentrations. It can be calculated from Figure 6 and Figure 7 that the Michaelis constant (Km) and the maximum reaction rate (Vmax) of Fe-NMPs for TMB are 0.367 mmol / L and 10.1×10 -8 mol / (L·s), respectively. The Michaelis constant of natural horseradish peroxidase (HRP) for TMB is 0.434 mmol / L, and the maximum reaction rate is 10×10 -8 mol / (L·s). When the maximum reaction rate of Fe-NMPs is the same as that of HRP, it has a smaller Km, indicating that Fe-NMPs have a better affinity for TMB compared to HRP.

[0069] (5) Evaluation of the affinity of iron-melanin for hydrogen peroxide

[0070] Add 10 μL of 0.2 mg / mL iron-melanin solution to 11 portions of sodium acetate-acetic acid buffer (pH = 3), then add different volumes of 20 mmol / L hydrogen peroxide and 10 μL of 20 mmol / L TMB solution. Make up the volume to 200 μL with sodium acetate-acetic acid buffer (pH = 3) so that the final concentrations of hydrogen peroxide in the 11 buffer systems are 0.1 mmol / L, 0.2 mmol / L, 0.4 mmol / L, 0.6 mmol / L, 0.8 mmol / L, 1.0 mmol / L, 1.2 mmol / L, 1.4 mmol / L, 1.6 mmol / L, 1.8 mmol / L, 2.0 mmol / L in turn. Measure the absorbance of the reaction system in each well at 652 nm every 7.5 s. The measurement results are as shown in Figure 8 and Figure 9 . Figure 8 Figure showing the fitting curve of the Michaelis kinetic constant of iron-melanin at different hydrogen peroxide concentrations, Figure 9 Figure showing the reaction rate of iron-melanin at different hydrogen peroxide concentrations. It can be calculated from Figure 9 that the maximum reaction rate of Fe-NMPs for hydrogen peroxide is 12×10 -8 mol / (L·s), while the maximum reaction rate of HRP for H2O2 is 8.71×10 -8 mol / (L·s). It can be seen that Fe-NMPs, as a peroxidase mimic enzyme, has a faster reaction rate than HRP, indicating that Fe-NMPs have more excellent peroxidase activity compared to HRP.

[0071] (6) Determination of the stability of iron-melanin peroxidase mimic enzyme

[0072] Add 10 μL of 0.2 mg / mL iron-melanin solution, 10 μL of 20 mmol / L TMB solution, and 10 μL of 20 mmol / L hydrogen peroxide to sodium acetate-acetic acid buffer (pH = 3), and make up the volume to 200 μL with sodium acetate-acetic acid buffer (pH = 3). Stir for 15 min to mix the buffer system evenly, and measure the absorbance of the buffer system at 652 nm. After the measurement, store the buffer system in the experimental environment (temperature 20 - 30 °C, humidity 45% - 80%), and measure the absorbance value of the buffer system at 652 nm every 5 days. The measurement results are as Figure 10 shown. It can be seen from Figure 10 that when the buffer system is directly placed in the laboratory without strictly controlling the storage environment, within 20 days, although the activity of the iron-melanin peroxidase mimetic enzyme gradually decreases, the decrease amplitude is not large, and there is still 90% of the reaction activity on the 20th day, indicating that the iron-melanin peroxidase mimetic enzyme prepared by the present invention has excellent stability under general experimental conditions.

[0073] (7) Determination of the reusability of the iron-melanin peroxidase mimetic enzyme

[0074] Add 10 μL of 0.2 mg / mL iron-melanin solution, 10 μL of 20 mmol / L TMB solution, and 10 μL of 20 mmol / L hydrogen peroxide to sodium acetate-acetic acid buffer (pH = 3), and make up the volume to 200 μL with sodium acetate-acetic acid buffer (pH = 3). Stir for 15 min to mix the buffer system evenly, and measure the absorbance of the buffer system at 652 nm. After the measurement, centrifuge the buffer system at 10000 r / min for 5 min to recover the iron-melanin, wash it with deionized water, and then reuse the obtained iron-melanin according to the aforementioned method and measure the absorbance value of the buffer system at 652 nm; repeat the cycle 5 times in total; the measurement results are as Figure 11 shown. It can be seen from Figure 11 that the catalytic activity of the iron-melanin prepared in the present invention decreases very little during recycling. After recycling 5 times, the activity is still more than 90%, indicating that the iron-melanin peroxidase mimetic enzyme prepared by the present invention has good reusability under general experimental conditions.

[0075] 3. Detection of total antioxidant capacity

[0076] As Figure 1 shown, the iron-melanin in the present invention can catalyze hydrogen peroxide to oxidize the chromogenic substrate TMB, and the oxidized chromogenic substrate can be reduced to TMB by ascorbic acid. Based on this principle, the total antioxidant capacity of the sample can be measured. The method for measuring the total antioxidant capacity of the sample in the present invention includes the following steps:

[0077] S1. Add 10 μL of a 20 mmol / L TMB solution, 10 μL of a 20 mmol / L hydrogen peroxide solution, and 10 μL of a 0.2 mg / mL iron-melanin solution to a sodium acetate - acetic acid buffer solution (pH = 3), and stir evenly.

[0078] S2. Prepare 22 portions of the buffer system in step S1. Add ascorbic acid solution to each portion, and make up to 200 μL with sodium acetate - acetic acid buffer solution (pH = 3), so that the concentrations of ascorbic acid in the final buffer systems are 0, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, 250 mmol / L in sequence. The buffer system without added ascorbic acid is used as the control group.

[0079] S3. Incubate the 22 portions of the buffer system in step S2 at 37 °C for 15 min, measure the ultraviolet - visible absorption spectrum in the range of 350 - 800 nm, and record the absorbance values as Y0, Y1, Y2... Y 21 ; Calculate the differences between Y1, Y2... Y 21 and Y0 respectively, conduct data analysis on the absorbance differences and antioxidant concentrations, establish the model relationship between the ascorbic acid concentration x and the absorbance difference Y, as shown in Figure 12 . The standard curve for detecting antioxidants obtained is Y = 0.00189x + 0.03456 (R 2 = 0.991); It can be calculated from Figure 12 that the lowest detection limit of iron-melanin as a peroxidase mimicking enzyme for detecting ascorbic acid is 12.61 μmol / L, and the detection range is 5 μmol / L - 250 μmol / L.

[0080] S4. Select vitamin C tablets produced by Northeast Pharmaceutical Group, vitamin C tablets produced by Conba, three vitamin C-containing beverages purchased from the supermarket, oranges and lemons purchased from the supermarket, and prepare sample solutions with concentrations within the detection range in step S3. Measure the absorbance values of each sample solution at 652 nm according to the methods in steps S1 - S3, substitute the difference between the absorbance value of the sample solution and the absorbance of the control group into the standard curve in step S3, and calculate the concentration of ascorbic acid in the sample solution. The results are as shown in Figure 13 and Figure 14As shown; the total antioxidant capacity (TAC) of all sample solutions was converted into millimolar equivalents of ascorbic acid (AA), and the TAC content in the samples was expressed in units of AA / L. Figure 13 It is a comparison chart of the results detected by using the method of the present invention for two vitamin C tablets and the content indicated in the specification. It can be seen from the figure that the results measured according to the method of the present invention have little difference from the content indicated in the specification, indicating that the method for measuring the total antioxidant capacity in the present invention is accurate and effective. Figure 14 It is the detection results of the total antioxidant capacity of three beverages, oranges and lemons. The detection results of the three beverages are basically consistent with the results on the beverage bottle labels. The TAC in the orange juice beverage is higher than that in the oranges, probably because some antioxidants are added during the beverage production process; the TAC of lemons is higher than that of oranges, indicating that the content of ascorbic acid in lemons is higher than that in oranges.

[0081] The above are only the preferred embodiments of the present invention and are not used to limit the protection scope of the present invention. For any person skilled in the art, various changes and modifications can be made to the present invention. Any simple equivalent changes and modifications made according to the protection scope of the present invention application and the content of the specification shall be included in the protection scope of the present invention.

Claims

1. Application of iron-melanin peroxidase mimicking enzyme, characterized in that, In the method of using iron-melanin as a peroxidase mimetic enzyme to detect the total antioxidant capacity; The preparation method of the iron-melanin includes the following steps: P1. Add squid ink into deionized water, stir until the squid ink is evenly suspended, wash the obtained suspension with deionized water and centrifuge 5-7 times, and freeze-dry to obtain melanin nanoparticles; P2. Resuspend the melanin nanoparticles in deionized water, add an aqueous solution containing Fe 3+ to obtain a mixed solution C, and adjust the pH of the mixed solution C to 3 with a pH regulator, followed by stirring and reacting for 6 h; the concentration of the melanin nanoparticles in the mixed solution C is 12.5 mg / mL; the concentration of Fe 3+ in the mixed solution C is 9.6 mmol / mL; P3. Centrifuge and wash the aggregate solution obtained after the reaction in step P2 with deionized water 5-7 times to obtain an iron-melanin solution, and freeze-dry to obtain iron-melanin powder; The method of using iron-melanin as a peroxidase mimetic enzyme to detect the total antioxidant capacity includes the following steps: S1. Add hydrogen peroxide, a chromogenic substrate, and iron-melanin to an acetate-sodium acetate buffer solution, stir evenly to obtain a mixed solution A. The concentration of hydrogen peroxide in the mixed solution A is 1.0 mmol / L, the concentration of the chromogenic substrate is 1.0 mmol / L, and 0 < the concentration of iron-melanin ≤ 50 μg / mL; S2. Take N portions of the mixed solution A, respectively add antioxidant aqueous solutions with different concentrations and mix evenly, stand and incubate to obtain N test solutions B with different antioxidant concentrations; the concentration of the antioxidant in the test solution B is 0-0.3 mmol / L, and the test solution B with an antioxidant concentration of 0 is used as a control group; S3. Measure the ultraviolet-visible absorption spectra of the N portions of the test solution B in step S2 within the range of 350 - 800 nm, and record the absorbance values of the control group and the other test solutions B at 652 nm as Y0, Y1, Y2... Y N-1 respectively; calculate the differences between Y1, Y2... Y N-1 and Y0, conduct data analysis on the absorbance differences and antioxidant concentrations, establish the model relationship between the antioxidant concentration x and the absorbance difference Y, and obtain the standard curve for detecting antioxidants as Y = 0.00189x + 0.03456; S4. Prepare a sample solution with an unknown antioxidant concentration according to the methods in steps S1 and S2, measure the absorbance value of the sample solution at 652 nm, substitute the difference between the obtained absorbance value and the absorbance value Y0 of the control group into the standard curve in step S3, and calculate the concentration of the antioxidant.

2. The application of the iron-melanin peroxidase mimic enzyme according to claim 1, wherein The pH of the acetate-sodium acetate buffer solution is 2-6.

3. Use of the iron-melanin peroxidase mimicking enzyme according to claim 1, characterized in that, The temperature for preparing the test solution B in step S2 is 35-70 °C.

4. The application of the iron-melanin peroxidase mimicking enzyme according to claim 1, wherein The standing incubation time in step S2 is 5-25 min.

5. The application of the iron-melanin peroxidase mimicking enzyme according to claim 1, wherein The chromogenic substrate is 3,3',5,5'-tetramethylbenzidine.

6. The application of the iron-melanin peroxidase mimicking enzyme according to claim 1, wherein The antioxidant is ascorbic acid.

7. Use of the iron-melanin peroxidase mimetic enzyme according to claim 1, characterized in that, The concentration of squid ink in the suspension in step P1 is 0.25 g / mL.

8. Use of the iron-melanin peroxidase mimetic enzyme according to claim 1, characterized in that, The aqueous solution containing Fe described in step P2 3+ is an aqueous solution of FeCl3.

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