CN114113066B - Application of maltol iron peroxide mimic enzyme in detecting hydrogen peroxide and total antioxidant capacity - Google Patents
Application of maltol iron peroxide mimic enzyme in detecting hydrogen peroxide and total antioxidant capacity Download PDFInfo
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Abstract
The invention relates to application of maltol iron peroxide mimic enzyme in detecting hydrogen peroxide and total antioxidant capacity, belonging to the technical field of detection and analysis. Iron maltol can catalyze H as a peroxide mimic enzyme 2 O 2 Decomposing to obtain active oxygen O 2 ·‑ ，O 2 ·‑ Can oxidize chromogenic substrate more effectively, and shows obvious absorption peak at 652nm of ultraviolet-visible absorption spectrum. Absorbance value is respectively related to H in the sample 2 O 2 The concentration or AA concentration shows good linear relation, so that the concentration or AA concentration can be used for realizing H by measuring the absorbance value of a sample 2 O 2 And rapid sensitive colorimetric detection of TAC equivalent to AA. H provided in the invention 2 O 2 The lowest detection limit of the detection method is 0.45 mu mol/L, and the lowest detection limit of the provided AA detection method is 0.86 mu mol/L.
Description
Technical Field
The invention belongs to the technical field of detection and analysis, and particularly relates to application of ferric maltol as a peroxide mimic enzyme in detecting hydrogen peroxide and total antioxidant capacity.
Background
Hydrogen peroxide (H) 2 O 2 ) Is an important oxide and relates to chemical, biological, pharmaceutical, clinical and environmental processes. H in human body 2 O 2 The content of (2) is closely related to human health. At the same time H 2 O 2 Due to their oxidative properties, they are often used as food preservatives and bactericides. Adding H into milk 2 O 2 Not only can inhibit the growth of bacteria and prevent deterioration, but also can improve the quality of dairy products by activating an internal lactoperoxidase system. However, H is used in excess 2 O 2 Can cause injury to the body, including duodenal diseases, neurodegenerative diseases, and diabetes. On the other hand, antioxidants are reducing agents that protect cells and organs from oxidative stress and prevent oxidative damage, and thus various antioxidants such as Ascorbic Acid (AA), gallic Acid (GA) and Tannic Acid (TA) have been widely used as additives in foods, medicines and cosmetics. Research shows that the dosage and effect of the antioxidant are closely related to the antioxidant capacity and the antioxidant mechanism. However, it is difficult to analyze the antioxidant capacity of individual antioxidants because of the wide variety of antioxidants and the possible interactions between them. Thus, the Total Antioxidant Capacity (TAC) was used to evaluate the sum of the antioxidant capacities of all antioxidants in the test sample.
Natural enzymes have high efficiency and specificity for substrates in biological systems, but natural enzymes suffer from inherent disadvantages of high cost, low stability and difficulty in storage, so that their use is limited. In recent years, various nano-enzymes have attracted attention from researchers due to their high stability and easy acquisition, and exhibit excellent colorimetric properties in the field of biosensors. The first nanoenzyme (Fe) with natural peroxidase mimic properties since 2007 3 O 4 ) Various nanoezymes have been reported to have a natural enzymatic role in succession. Wherein the peroxidase, such as horseradish peroxidase (HRP), can be used as a smart biocatalyst for H 2 O 2 And (5) detecting. In addition, a peroxidase-like nanometer is also constructedEnzyme detection of bioactive small molecules (e.g., H 2 O 2 Glucose and AA), metal ions (e.g. Ag + 、Hg 2+ And Ce (Ce) 3+ ) Nucleic acids, proteins, and even viruses. These peroxidase-like nanomaterials have significant advantages over the natural enzymes, including good operational stability, high catalytic activity, low synthesis and purification costs, and good recyclability.
Chinese patent application CN113617395A discloses a nano enzyme for detecting food antioxidant activity and a preparation method thereof, wherein potassium hexacyanoferrate and citric acid are dissolved in water, then sodium tetrachloroaurate (III) and copper sulfate are sequentially added, and gold doped nano enzyme Au@Cu-HCF is obtained through reaction. The nano-enzyme can be used for detecting the hydrogen atom transfer type antioxidant substances and can also be used for detecting the single electron transfer type antioxidant substances. However, noble metal-based nanoezymes, although having high peroxidase activity, have limited their wide application due to their high price and scarcity.
Thus, there is an urgent need to find artificial peroxidase mimic enzymes with high activity, controlled synthesis, low cost, and excellent stability under stringent conditions to circumvent the drawbacks of natural enzymes.
Disclosure of Invention
In order to solve the problems, the invention uses ferric maltol as a peroxide mimic enzyme to catalyze hydrogen peroxide to oxidize chromogenic substrates so as to realize H 2 O 2 The sensitive and rapid detection of the total oxidation resistance is realized by utilizing the action mechanism that the antioxidant can reduce the oxidized chromogenic substrate in an oxidation system.
To achieve the first object described above, a method for detecting hydrogen peroxide using iron maltol as a peroxide mimic enzyme according to the present invention comprises the steps of:
s1, adding ferric maltol and a chromogenic substrate into an acetate buffer solution to obtain a first mixed solution, wherein the total volume of the first mixed solution is 200 mu L, the final concentration of the ferric maltol is 20 mu g/mL, and the final concentration of the chromogenic substrate is 2mmol/L;
s2, taking N parts of the first mixed solution in the step S1, respectively adding hydrogen peroxide solutions with different concentrations to obtain N parts of to-be-detected solution A with different hydrogen peroxide concentrations, and uniformly stirring; the concentration range of hydrogen peroxide in the solution A to be detected is 1-100 mu mol/L;
s3, measuring the absorbance value of N parts of the liquid A to be measured in the step S2 at 625nm of the ultraviolet-visible absorption spectrum, and marking as y 1 The method comprises the steps of carrying out a first treatment on the surface of the For absorbance value y 1 And hydrogen peroxide concentration for data analysis to establish hydrogen peroxide concentration and absorbance value y 1 The model relation between the two is used for obtaining the standard curve y for detecting the hydrogen peroxide 1 ＝0.0121x 1 +0.4296, where x 1 Is hydrogen peroxide concentration;
s4, preparing a solution with unknown hydrogen peroxide concentration according to the method in the steps S1 and S2, measuring the absorbance value of the solution at 625nm of the ultraviolet-visible absorption spectrum, substituting the obtained absorbance value into the standard curve in the step S3, and calculating the hydrogen peroxide concentration.
Preferably, in step S1, the pH of the acetate buffer is=2 to 5.
Further preferably, in step S1, the pH of the acetate buffer is=2 to 3.
Preferably, the chromogenic substrate in step S1 is 3,3', 5' -tetramethylbenzidine.
Preferably, the temperature of the solution to be measured a prepared in step S2 is 20 to 60 ℃.
Further preferably, the temperature of the solution to be measured a prepared in step S2 is 35 to 55 ℃.
Still more preferably, the temperature of the solution to be measured a prepared in step S2 is 40 to 50 ℃.
Still more preferably, the temperature of the solution to be measured a prepared in step S2 is 40 to 45 ℃.
Still more preferably, the temperature of the solution to be measured a prepared in step S2 is 40 ℃.
To achieve the second object described above, a method for detecting total antioxidant capacity using iron maltol as a peroxide mimic enzyme according to the present invention comprises the steps of:
p1, adding ferric maltol, a chromogenic substrate and hydrogen peroxide into an acetate buffer solution to obtain a second mixed solution, wherein the total volume of the second mixed solution is 200 mu L, the final concentration of the ferric maltol is 20 mu g/mL, the final concentration of the chromogenic substrate is 2mmol/L, and the final concentration of hydrogen peroxide is 50 mu mol/L;
p2, taking M parts of the second mixed solution in the step P1, respectively adding antioxidant water solutions with different concentrations to obtain M parts of to-be-detected solution B with different antioxidant concentrations, and uniformly stirring; the concentration range of the antioxidant in the liquid B to be detected is 1-40 mu mol/L; taking the mixed solution without the antioxidant in the step P1 as a control group;
p3, measuring absorbance values of the control group and M parts of the liquid B to be measured in the step P2 at 625nm of ultraviolet-visible absorption spectrum, respectively marked as Y 1 And Y 2 Y is taken as 1 And Y is equal to 2 The difference in (2) is recorded as y 2 The method comprises the steps of carrying out a first treatment on the surface of the For absorbance difference y 2 And the antioxidant concentration is subjected to data analysis, and an antioxidant concentration and absorbance difference y are established 2 The model relation between the antioxidant and the antioxidant is obtained to obtain a standard curve y of the antioxidant 2 ＝0.0148x 2 -0.0068, wherein x 2 Is the antioxidant concentration;
p4, preparing a solution with unknown antioxidant concentration according to the method in the steps P1 and P2, measuring the absorbance value of the solution at 625nm of the ultraviolet-visible absorption spectrum, substituting the difference value between the obtained absorbance value and the absorbance value of the control group into the standard curve of the step P3, and calculating the concentration of the antioxidant.
Preferably, in step P1, the pH of the acetate buffer is=2 to 5.
Further preferably, in step P1, the pH of the acetate buffer is=2 to 3.
Preferably, the chromogenic substrate in step P1 is 3,3', 5' -tetramethylbenzidine.
Preferably, the temperature of the solution B to be measured prepared in the step P2 is 20-60 ℃.
Further preferably, the temperature of the solution B to be measured prepared in the step P2 is 35-55 ℃.
Still more preferably, the temperature of the solution B to be measured prepared in the step S2 is 40 to 50 ℃.
Still more preferably, the temperature of the solution B to be measured prepared in the step S2 is 40 to 45 ℃.
Still more preferably, the temperature of the solution B to be measured prepared in the step S2 is 40 ℃.
Preferably, the antioxidant is ascorbic acid.
Compared with the prior art, the invention has the beneficial effects that:
(1) The powerful metal chelating ability of the maltol provides an ultrafine catalytic active center for iron, shows good catalytic ability, and has the advantages of low cost, high stability and good biocompatibility; the iron maltol can be used as peroxide mimic enzyme to efficiently catalyze H 2 O 2 The active oxygen with strong oxidizing property is obtained by decomposition, the rapid oxidation of the chromogenic substrate can be realized, and an obvious absorption peak is shown at 652nm of an ultraviolet-visible absorption spectrum; by establishing a linear equation between the absorbance value of the oxidized chromogenic substrate and the concentration of hydrogen peroxide, the concentration of hydrogen peroxide can be rapidly, sensitively and accurately detected, the time required by the whole detection reaction is 10min, and the minimum detection limit (3 sigma/N) is 0.45 mu mol/L;
(2) Catalytic H using ferric maltol as a peroxide mimic enzyme 2 O 2 The oxidation chromogenic substrate and the action principle that the oxidized chromogenic substrate can be reduced by an antioxidant are established, a linear equation between the concentration of the antioxidant and the absorbance value of the oxidized chromogenic substrate is established, and the total antioxidant capacity of a sample to be detected can be rapidly, sensitively and accurately detected; in the sample containing ascorbic acid as the main antioxidant component, the lowest detection limit (3σ/N) of the ascorbic acid detected by the method of the present invention was 0.86 μmol/L, with the antioxidant capacity of ascorbic acid equivalent to the total antioxidant capacity.
Drawings
FIG. 1 shows the use of maltol iron peroxide mimetic enzymes for the detection of H 2 O 2 And a schematic diagram of the overall antioxidant capacity;
FIG. 2 shows the presence or absence of H in the case of maltol iron peroxide mimetic enzyme 2 O 2 In the presence of H 2 O 2 Oxidizing TMB and H in the presence of 2 O 2 Spectral scan curves of TMB alone;
FIG. 3 shows the catalysis of H by maltol iron peroxide mimetic enzymes at different pH values 2 O 2 Absorbance versus activity intensity (%) histogram of oxidized TMB;
FIG. 4 shows the catalysis of H by maltol iron peroxide mimetic enzymes at different temperatures 2 O 2 Absorbance versus activity intensity (%) histogram of oxidized TMB;
FIG. 5 shows the catalysis of iron maltol peroxide mimic enzyme to different concentrations of H 2 O 2 A spectral scan pattern of an oxidation chromogenic substrate for color development;
FIG. 6 shows the absorbance at 652nm of the oxidized chromogenic substrate with H 2 O 2 Concentration linear standard curve graph;
FIG. 7 is a schematic illustration of the iron maltol peroxide mimic enzyme catalysis H 2 O 2 Adding ascorbic acid with different concentrations after oxidizing the chromogenic substrate to scan a spectrum chart;
FIG. 8 is a linear standard graph of absorbance at 652nm versus ascorbic acid concentration for a chromogenic substrate after oxidation;
FIG. 9 is a schematic illustration of the iron maltol peroxide mimic enzyme catalysis H 2 O 2 Generating a species verification map of active oxygen;
FIG. 10 is a graph showing the results of measuring the total antioxidant capacity of orange juice and sea buckthorn juice manufactured by Yang Ling Cyclo-horticultural Co., ltd according to the method provided by the present invention;
FIG. 11 is a graph showing the comparison of the total antioxidant capacity of farmer mountain spring vitamin water and farmer mountain spring water-soluble C100 and the actual total antioxidant capacity of farmer mountain spring water according to the method provided by the invention.
Detailed Description
The following description of the embodiments of the present invention will be made more apparent and fully by reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. Any equivalent alterations or substitutions by those skilled in the art based on the following embodiments are within the scope of the present invention.
Examples
1. Maltol iron peroxide mimic enzyme catalysis H 2 O 2 Activity determination of (A)
Malt phenol iron peroxide mimic enzyme catalyzed H was performed as follows 2 O 2 Activity determination of (2):
control group (H) 2 O 2 +tmb): taking 20 mu L of 0.5mmol/L hydrogen peroxide solution and 20 mu L of 20mmol/L TMB (3, 3', 5' -tetramethyl benzidine) solution, and placing the solution in acetate buffer with pH=3 to ensure that the volume of the total system is 200 mu L;
no H 2 O 2 Group (fm+tmb): preparing Ferric Maltol (FM) into an aqueous solution with the concentration of 200 mu g/mL, taking 20 mu L of the ferric maltol aqueous solution, adding 20 mu L of a TMB solution with the concentration of 20mmol/L, and adding the TMB solution into acetate buffer with the pH of=3 to ensure that the volume of the total system is 200 mu L;
with H 2 O 2 Group (FM+H) 2 O 2 +tmb): taking 20 mu L of 200 mu g/mL maltol molten iron solution, adding 20 mu L of 500 mu mol/L hydrogen peroxide solution, adding the mixed solution into acetate buffer solution with pH value of 3 to prepare the maltol molten iron with the concentration of 20 mu g/mL and H 2 O 2 The solution at a concentration of 50. Mu. Mol/L was continued to add 20. Mu.L of a 20mmol/L TMB solution and the total system volume was 200. Mu.L. The iron maltol used in the experiment was purchased from Beijing Happy biotechnology Co. The inventors found during the experiment that the smaller the particle size of iron maltol, the higher its catalytic activity as a peroxide mimetic enzyme.
And respectively standing and incubating the three groups of experimental solutions for 10min, and then measuring the absorbance of the reaction system at 400-800 nm in each hole of each group of experimental solutions by using an enzyme-labeled instrument (model: austria Tecan Austria GmbH). According to the above H 2 O 2 The same preparation method of the group prepares experimental solutions with pH values of 2, 3, 4, 6, 7 and 8 respectively, and uses an enzyme-labeled instrument (model: austria Tecan Austria GmbH) to measure the absorbance of the reaction system at 400-800 nm in each hole of each pH system experimental solution after standing and incubating for 10min respectively,the measurement results are shown in FIGS. 2 and 3. H is visible to naked eyes in the standing incubation process 2 O 2 The chromogenic substrate TMB of the group appears blue, indicating that ferric maltol is capable of catalyzing H 2 O 2 Reactive Oxygen Species (ROS) are decomposed. From FIG. 2, it can be seen that the iron maltol nano-mimic enzyme can catalyze H 2 O 2 Further, TMB was oxidized to generate a distinct absorption peak at 652 nm. As can be seen from fig. 3, when the pH of the reaction system is between 2 and 6, the iron maltol has the activity of peroxide mimic enzyme, but the activity is very low at ph=6, and the activity can reach 100% at ph=2 to 3.
50. Mu.L of 200. Mu.g/mL of ferric maltol peroxide mimic enzyme and 50. Mu.L of 0.5mmol/L H 2 O 2 To 20mmol/L acetate buffer (pH=3), 50. Mu.L of a 20mmol/L TMB solution was added thereto, and the mixture was fixed to a volume of 500. Mu.L with pH=3 acetate buffer. The mixed solution is placed in a metal heater, and is heated, bathed and incubated for reaction for 10min under the conditions that the temperature gradient is 5 ℃ and the temperature is 20-60 ℃, and an enzyme-labeled instrument is used for measuring the absorbance value of the mixed solution at 652nm at each temperature. All assays were performed in triplicate and the results of the assays are shown in figure 4. As can be seen from FIG. 4, when the pH value of the reaction system is 4 and the temperature is 20-60 ℃, the iron maltol shows good peroxide mimic enzyme catalytic activity, and particularly, the catalytic activity reaches more than 80% under the condition of 35-55 ℃; the catalytic activity reaches 100% at 40 ℃. For the convenience of the experiment, ph=3, 25 ℃ was chosen as the assay condition for the study.
2. Detection of hydrogen peroxide using iron maltol as peroxide mimic enzyme
The method for detecting hydrogen peroxide in the invention comprises the following steps:
s1, adding ferric maltol and TMB into acetate buffer solution with pH=3 to obtain a first mixed solution, wherein the total volume of the first mixed solution is 200 mu L, the final concentration of the ferric maltol is 20 mu g/mL, and the final concentration of the TMB is 2mmol/L;
s2, taking 10 parts of the first mixed solution obtained in the step S1, respectively adding hydrogen peroxide solutions with different concentrations to obtain 10 parts of to-be-detected solution A with different hydrogen peroxide concentrations, stirring, and stabilizing for 10 minutes; the concentration of hydrogen peroxide in 10 parts of the solution A to be tested is sequentially 1 mu mol/L, 10 mu mol/L, 20 mu mol/L, 30 mu mol/L, 40 mu mol/L, 50 mu mol/L, 60 mu mol/L, 70 mu mol/L, 80 mu mol/L, 90 mu mol/L and 100 mu mol/L;
s3, measuring the absorbance value of 10 parts of the liquid A to be measured in the step S2 at 625nm of the ultraviolet-visible absorption spectrum, and marking as y 1 (the measurement results are shown in FIG. 5); for absorbance value y 1 And hydrogen peroxide concentration for data analysis to establish hydrogen peroxide concentration and absorbance value y 1 The model relation between the two is obtained to obtain a standard curve (shown in figure 6) of the detected hydrogen peroxide as y 1 ＝0.0121x 1 +0.4296(R 2 =0.995), where x 1 Is hydrogen peroxide concentration; the minimum detection limit (3σ/N) calculated from the standard curve is 0.45 μmol/L;
s4, preparing a solution with unknown hydrogen peroxide concentration according to the method in the steps S1 and S2, measuring the absorbance value of the solution at 625nm of the ultraviolet-visible absorption spectrum, substituting the obtained absorbance value into the standard curve in the step S3, and calculating the hydrogen peroxide concentration.
As can be seen from fig. 5, as the hydrogen peroxide concentration increases, the absorbance value of the liquid a to be measured at 652nm also increases, which indicates that under the catalysis of the maltol iron peroxide mimic enzyme, TMB can be effectively oxidized, an obvious absorption peak appears at 652nm, and the larger the hydrogen peroxide concentration of the catalytic substrate is, the more TMB is oxidized, and the higher the absorbance value is; as can be seen from fig. 6, the absorbance value of the solution a to be measured at 652nm shows a good linear relationship with the concentration of hydrogen peroxide, which means that the concentration of hydrogen peroxide in the sample can be calculated from the absorbance value of the measured sample so as to quantitatively analyze the hydrogen peroxide.
3. Iron maltol as peroxide mimic enzyme to detect total antioxidant capacity
The method for detecting the total antioxidant capacity by using the antioxidant capacity equivalent total antioxidant capacity of the ascorbic acid (vitamin C, VC for short) comprises the following steps:
p1, adding ferric maltol, TMB and hydrogen peroxide into acetate buffer solution with pH=3 to obtain a second mixed solution, wherein the total volume of the second mixed solution is 200 mu L, the final concentration of the ferric maltol is 20 mu g/mL, the final concentration of the TMB is 2mmol/L, and the final concentration of the hydrogen peroxide is 50 mu mol/L;
p2, taking 10 parts of the second mixed solution obtained in the step P1, respectively adding ascorbic acid water solutions with different concentrations to obtain 10 parts of to-be-detected solution B with different ascorbic acid concentrations, stirring, and stabilizing for 10 minutes; the concentration of the ascorbic acid in 10 parts of the liquid B to be detected is sequentially 1 mu mol/L, 2 mu mol/L, 5 mu mol/L, 10 mu mol/L, 15 mu mol/L, 20 mu mol/L, 25 mu mol/L, 30 mu mol/L, 35 mu mol/L and 40 mu mol/L; taking the mixed solution without the antioxidant in the step P1 as a control group;
p3, the absorbance values of the control group and 10 parts of the liquid B to be detected in the measurement step P2 at 625nm of the ultraviolet-visible absorption spectrum are respectively marked as Y 1 And Y 2 (as shown in FIG. 7), Y 1 And Y is equal to 2 The difference in (2) is recorded as y 2 The method comprises the steps of carrying out a first treatment on the surface of the For absorbance difference y 2 And ascorbic acid concentration, and establishing an absorbance difference y 2 The model relation between the ascorbic acid and the ascorbic acid is obtained to obtain a standard curve (shown in figure 8) of y 2 ＝0.0148x 2 -0.0068(R 2 =0.995), where x 2 Is the concentration of ascorbic acid; the minimum detection limit (3σ/N) calculated from the standard curve was 0.86 μmol/L;
and P4, preparing a solution with unknown ascorbic acid concentration according to the method in the steps P1 and P2, measuring the absorbance value of the solution at 625nm of the ultraviolet-visible absorption spectrum, substituting the difference value between the obtained absorbance value and the absorbance value of the control group into the standard curve of the step P3, and calculating the concentration of the ascorbic acid.
As can be seen from fig. 7, as the concentration of ascorbic acid increases, the absorbance value of the test solution B at 652nm decreases, which means that as ascorbic acid is added, TMB oxidized by hydrogen peroxide is reduced to TMB by ascorbic acid again, and the greater the concentration of ascorbic acid, the more TMB is reduced; as can be seen from fig. 8, the absorbance value of the test solution B at 652nm shows a good linear relationship with the ascorbic acid concentration, which indicates that the concentration of the ascorbic acid in the sample can be calculated from the absorbance value of the measured sample, so as to quantitatively analyze the ascorbic acid to determine the antioxidant capacity of the sample.
4. Iron maltol catalysis H 2 O 2 ROS species validation by oxidation of TMB
10mmol/L of p-benzoquinone (PBQ), 10wt% of isopropyl alcohol (IPA) and 10wt% of tert-butyl alcohol (TBA), 10mmol/L of sodium azide (NaN) 3 ) Respectively as O 2 ·- OH, OH and 1 O 2 is a scavenger of (a). Respectively taking 50 mu L of four scavengers, sequentially adding ferric maltol solution and hydrogen peroxide, fixing the volume to 450 mu L by acetate buffer solution with pH=3, incubating for 3min, adding 50 mu L of TMB solution with 20mmol/L, wherein the final concentration of ferric maltol in each reaction system is 20 mu g/mL, and H 2 O 2 The concentration was 50. Mu. Mol/L and the final TMB concentration was 2mmol/L. Adding ferric maltol solution, hydrogen peroxide and TMB into acetate buffer with pH=3, and fixing volume to 500 μl with acetate buffer with pH=3 as control group, wherein final concentration of ferric maltol in control group is 20 μg/mL, H 2 O 2 The concentration was 50. Mu. Mol/L and the final TMB concentration was 2mmol/L. The absorbance at 652nm was measured for the control group and each reaction system. All assays were performed in triplicate and the results of the assays are shown in FIG. 9, as can be seen from FIG. 9, when PBQ was added as O 2 ·- Maltol iron peroxide mimic enzyme catalyzed H when scavenger is used 2 O 2 The ability to oxidize TMB was significantly reduced (the amount of absorbance change was significantly increased), while the addition of other scavengers was not effective in inhibiting the iron maltol peroxide mimic enzyme from catalyzing H 2 O 2 The oxidation effect on TMB is further proved to be O as the effective active oxygen generated in the catalytic process 2 ·- 。
From all the above experiments, the inventors have concluded that iron maltol peroxide mimic enzyme is capable of catalyzing H as a peroxide mimic enzyme in the principle of detecting hydrogen peroxide and total antioxidant capacity as shown in FIG. 1 2 O 2 Decomposing to obtain active oxygen O 2 ·- ，O 2 ·- TMB can be oxidized more effectively, and obvious absorption peak is shown at 652 nm. Absorbance values are respectivelyH 2 O 2 The concentration or AA concentration shows good linear relation, so that the concentration or AA concentration can be used for realizing H by measuring the absorbance value of a sample 2 O 2 And rapid sensitive colorimetric detection of TAC equivalent to AA.
5. Practical application of ferric maltol as peroxide mimic enzyme to detect total antioxidant capacity
Orange juice and sea buckthorn juice produced by Yang Ling Cyclo horticulture Limited which are purchased in supermarket are respectively centrifuged for 10min at 10000r/min at 4 ℃ to remove sediment, the supernatant is diluted by ultrapure water to 100 times until the concentration is within a detection range (1-40 mu mol/L), and the obtained orange juice sample and sea buckthorn juice sample are stored in a refrigerator at 4 ℃ for standby. Preparing a second mixed solution according to the step P1, taking two parts of the second mixed solution, respectively adding 20 mu L of an orange juice sample and a sea buckthorn juice sample to obtain an orange juice to-be-detected solution and a sea buckthorn juice to-be-detected solution, uniformly stirring, measuring absorbance values of the two parts of to-be-detected solutions at 652nm, and calculating the AA content according to an established standard curve; each of the liquids to be measured was measured three times in parallel, and the average value of the three measurement results was taken, and the measurement results are shown in fig. 10. TAC is described as milliequivalents AA/L and as can be seen in FIG. 10, the TAC of the orange juice sample measured according to the method of the present invention is 51.78AA/L and the TAC of the sea buckthorn juice sample measured according to the method of the present invention is 48.92AA/L; the method of the invention can be used for measuring the total antioxidant capacity of daily foods or beverages with VC as a main antioxidant ingredient.
Diluting the VC-enriched farmer spring water solution C100 and farmer spring vitamin water purchased from supermarket with ultrapure water until the VC concentration is within the detection range, and performing no other treatment. Preparing a second mixed solution according to the step P1, taking two parts of the second mixed solution, respectively adding 20 mu L of diluted water-soluble C100 and vitamin water to obtain water-soluble C100 to-be-measured solution and vitamin water to-be-measured solution, uniformly stirring, measuring absorbance values of the two parts of to-be-measured solution at 652nm, and calculating the AA content according to an established standard curve; each of the liquids to be measured was measured three times in parallel, and the average value of the three measurement results was taken, and the measurement results are shown in fig. 11. TAC is described as milliequivalents AA/L, and as can be seen from FIG. 11, the TAC of the farmer mountain spring water-soluble C100 measured according to the method of the invention is 224.37AA/L, the calculated TAC according to the VC content on the batching table is 225AA/L, and the difference is 0.28%; the TAC of the farmer mountain spring vitamin water measured by the method is 198.38AA/L, the calculated TAC according to the VC content on the batching table is 200AA/L, and the difference is 0.81%.
By measuring the total antioxidant capacity of the two different samples by the method of the invention and comparing the measurement result with the data converted according to the VC content on the sample label ingredients, the total antioxidant capacity of the sample measured by the method of the invention is the same as the actual total antioxidant capacity of the sample, and the standard curve established in the invention can be accurately and rapidly used for measuring the total antioxidant capacity of the sample.
Although the above examples are described with respect to the total antioxidant capacity of a beverage, the method of the present invention is not limited to the measurement of the total antioxidant capacity of a food or beverage, and the method of the present invention may be used to measure the total antioxidant capacity of a sample to be measured as long as VC is the main antioxidant component in the sample to be measured.
The foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Various modifications and alterations of this invention will occur to those skilled in the art. Any and all such simple and equivalent variations and modifications are intended to be included within the scope of this invention.
Claims (4)
1. Use of ferric maltol peroxide mimic enzyme, characterized in that ferric maltol is used as a peroxide mimic enzyme in a method for detecting hydrogen peroxide and/or total antioxidant capacity; wherein, the method for detecting hydrogen peroxide by using the maltol iron as the peroxide mimic enzyme comprises the following steps:
s1, adding ferric maltol and a chromogenic substrate into acetate buffer solution with pH=2 to obtain a first mixed solution, wherein the total volume of the first mixed solution is 200 mu L, the final concentration of the ferric maltol is 20 mu g/mL, and the final concentration of the chromogenic substrate is 2mmol/L;
s2, taking N parts of the first mixed solution in the step S1, respectively adding hydrogen peroxide solutions with different concentrations to obtain N parts of to-be-detected solution A with different hydrogen peroxide concentrations, and uniformly stirring; the concentration range of hydrogen peroxide in the solution A to be detected is 1-100 mu mol/L; the temperature of the liquid A to be detected is 35-55 ℃;
s3, measuring absorbance values of N parts of the liquid A to be measured in the step S2 at 625nm of an ultraviolet-visible absorption spectrum, and marking the absorbance values as y1; data analysis is carried out on the absorbance value y1 and the hydrogen peroxide concentration, and a model relation between the hydrogen peroxide concentration and the absorbance value y1 is established, so that a standard curve for detecting hydrogen peroxide is obtained as y1=0.0121×1+0.4296, wherein x1 is the hydrogen peroxide concentration;
s4, preparing a solution with unknown hydrogen peroxide concentration according to the method in the steps S1 and S2, measuring the absorbance value of the solution at 625nm of the ultraviolet-visible absorption spectrum, substituting the obtained absorbance value into the standard curve of the step S3, and calculating the hydrogen peroxide concentration;
the method for using ferric maltol as a peroxide mimic enzyme for detecting total antioxidant capacity comprises the following steps:
p1, adding ferric maltol, a chromogenic substrate and hydrogen peroxide into an acetate buffer solution with pH=2 to obtain a second mixed solution, wherein the total volume of the second mixed solution is 200 mu L, the final concentration of the ferric maltol is 20 mu g/mL, the final concentration of the chromogenic substrate is 2mmol/L, and the final concentration of the hydrogen peroxide is 50 mu mol/L;
p2, taking M parts of the second mixed solution in the step P1, respectively adding antioxidant water solutions with different concentrations to obtain M parts of to-be-detected solution B with different antioxidant concentrations, and uniformly stirring; the concentration range of the antioxidant in the liquid B to be detected is 1-40 mu mol/L; taking the mixed solution without the antioxidant in the step P1 as a control group; the temperature of the liquid B to be detected is 35-55 ℃;
p3, measuring absorbance values of the control group and M parts of to-be-measured liquid B at 625nm of an ultraviolet-visible absorption spectrum in the step P2, respectively marking as Y1 and Y2, and marking the difference value between Y1 and Y2 as Y2; data analysis is carried out on the absorbance difference value y2 and the antioxidant concentration, and a model relation between the antioxidant concentration and the absorbance difference value y2 is established, so that a standard curve for detecting the antioxidant is obtained as y2=0.0148x2-0.0068, wherein x2 is the antioxidant concentration;
p4, preparing a solution with unknown antioxidant concentration according to the method in the steps P1 and P2, measuring the absorbance value of the solution at 625nm of the ultraviolet-visible absorption spectrum, substituting the difference value between the obtained absorbance value and the absorbance value of the control group into the standard curve of the step P3, and calculating the concentration of the antioxidant.
2. The use of an iron maltol peroxide-mimetic enzyme according to claim 1, characterized in that the chromogenic substrate in step S1 is 3,3', 5' -tetramethylbenzidine.
3. The use of an iron maltol peroxide-mimetic enzyme according to claim 1, characterized in that the chromogenic substrate in step P1 is 3,3', 5' -tetramethylbenzidine.
4. The use of an iron maltol peroxide mimetic enzyme as claimed in claim 1, wherein the antioxidant is ascorbic acid.
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