CN113912138A - High-efficiency detection of ascorbic acid mimic enzyme and preparation method thereof - Google Patents
High-efficiency detection of ascorbic acid mimic enzyme and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an ascorbic acid mimic enzyme for efficient detection and a preparation method thereof. Fe-Ni bimetallic sulfide Fe by hydrothermal method0.8Ni0.2S2Compounding with Graphene (GO) to synthesize Fe with lamellar structure and peroxidase-like activity0.8Ni0.2S2GO mimic enzyme, iron nickel sulfide Fe0.8Ni0.2S2Uniformly dispersed on the graphene sheet layer. The introduction of the graphene can effectively prevent Fe0.8Ni0.2S2The aggregation of the nano particles changes the contents of Fe and Ni with different oxidation numbers, increases the electron transfer and improves the enzyme activity. Fe0.8Ni0.2S2the/GO mimic enzyme can be used for detecting ascorbic acid at room temperature, and has short detection time and high selectivity. Fe0.8Ni0.2S2The preparation method of the/GO mimic enzyme is simple and convenient, and the needed raw materials have wide sources and low price.
Description
Technical Field
The invention relates to a mimic enzyme and a preparation method thereof, in particular to a mimic enzyme for efficiently detecting ascorbic acid and a preparation method thereof.
Background
Ascorbic Acid (AA) is a water-soluble vitamin, and is widely used as a preservative in foods, cosmetics and pharmaceuticals due to its good antioxidant activity. However, excessive amounts of AA can induce many diseases such as cold, mental illness, atherosclerosis, scurvy, cancer, etc., and thus there is a need for convenient and rapid detection of AA. The current AA detection method comprises an oxidant titration method, electrochemical detection, chromatographic analysis, fluorescence detection and a colorimetric method. The colorimetric method is based on the relation between the color of a substance and light absorption, the content of the substance to be detected is visually shown through the change of the color of a reactant, the sensitivity is high, the cost is low, and the colorimetric method has the characteristics of visualization, but the detection temperature is higher than the room temperature, such as 35-60 ℃, and the detection time is longer. To avoid these inconveniences, it is necessary to increase the activity of the mimetic enzyme used in the colorimetric method. Transition Metal Sulfides (TMSs) as nanoenzymes have good peroxidase-like activity, but the TMSs have poor dispersibility and low conductivity, so that the enzymatic activity is reduced, and the nanoenzymes have low affinity with hydrogen peroxide, so that high-concentration hydrogen peroxide is required in the determination process, the cost is increased, and environmental pollution is caused.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a method for efficiently detecting ascorbic acid mimic enzyme, which improves the dispersibility and the conductivity of metal sulfide, thereby improving the activity of the mimic enzyme and efficiently and quickly detecting ascorbic acid at room temperature; another objective of the invention is to provide a preparation method of the mimic enzyme.
The technical scheme is as follows: the invention relates to a high-efficiency detection ascorbic acid mimic enzyme, which comprises iron-nickel sulfide Fe0.8Ni0.2S2And graphene, iron nickel sulfide Fe0.8Ni0.2S2Uniformly dispersed on the graphene sheet layer.
The mimic enzyme is a lamellar two-dimensional structure.
The mass fraction of the mimic enzyme containing graphene is 5-15%.
The preparation method for efficiently detecting the ascorbic acid mimic enzyme comprises the following steps:
(1) mixing Fe (NO)3)3·9H2O、Ni(NO3)2·6H2O and Na2S·9H2Dissolving O in water, and adding a graphene aqueous solution containing sodium dodecyl benzene sulfonate to obtain a mixed solution;
(2) reacting the mixed solution at a constant temperature of 100-150 ℃ for 10-20 hours;
(3) and (3) cooling the reacted mixed material obtained in the step (2) to room temperature, performing centrifugal separation to obtain a solid product, alternately cleaning the solid product by using deionized water and ethanol, and finally performing vacuum drying at the temperature of 50-80 ℃ for 20-30 hours to obtain the mimic enzyme.
Said step (1) Fe (NO)3)3·9H2O、Ni(NO3)2·6H2O and Na2S·9H2The molar ratio of O is 9:1: 5-20, and the mass of water is 6-7 times of the mass of solid.
The mass ratio of the graphene to the sodium dodecyl benzene sulfonate to the water in the step (1) is 120-150: 5-8: 10000.
Said step (1) Fe (NO)3)3·9H2The mass ratio of O to graphene is 303: 5 to 25.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: (1) the graphene in the mimic enzyme can effectively prevent Fe0.8Ni0.2S2The aggregation of the nano particles changes the contents of Fe and Ni with different oxidation numbers, enhances the electron transfer and improves the enzyme activity; (2) the mimic enzyme can be used for detecting ascorbic acid at room temperature, has short detection time and high selectivity, can reduce the practical application cost, and has stronger practical application potential; (3) the preparation method of the mimic enzyme is simple and convenient, the source of the required raw materials is wide, the price is low, and the cost is saved.
Drawings
FIG. 1 shows Fe prepared in example 1 of the present invention0.8Ni0.2S2X-ray diffraction pattern of/GO;
FIG. 2 shows Fe prepared in example 1 of the present invention0.8Ni0.2S2Transmission electron microscopy of/GO;
FIG. 3 shows Fe prepared in example 1 of the present invention0.8Ni0.2S2EDX-mapping diagram of/GO;
FIG. 4 shows Fe prepared in example 1 of the present invention0.8Ni0.2S2Photoelectron energy spectrum of/GO;
FIG. 5 is a graph showing the results of experimental tests for peroxidase activity of different classes;
FIG. 6 shows Fe prepared in example 1 of the present invention0.8Ni0.2S2A graph of enzyme-like activity test results for/GO;
FIG. 7 shows Fe prepared in example 1 of the present invention0.8Ni0.2S2the/GO is used for a hydrogen peroxide detection result graph;
FIG. 8 shows Fe prepared in example 1 of the present invention0.8Ni0.2S2The result chart of the ascorbic acid detection by the/GO is shown.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
Example 1
(1) Preparation of mimic enzyme: 3.636g Fe (NO) were weighed out3)3·9H2O、0.291g Ni(NO3)2·6H2O (molar ratio of iron to nickel is 9:1) is added into 40mL of deionized water, ultrasonic dissolution is carried out, and then 2.400g of Na is added2S·9H2And O, continuously carrying out ultrasonic dissolution and uniformly mixing. 0.135g of graphene is added into 10ml of deionized water to obtain a suspension, 6.8 mg of sodium dodecyl benzene sulfonate is added, and the mixture is uniformly mixed. The two prepared solutions are mixed and transferred into a 100mL polytetrafluoroethylene reaction kettle, the reaction temperature is 110 ℃, and the reaction time is 12 hours. And after the reaction is finished, naturally cooling the reacted mixture to room temperature, and performing centrifugal separation to obtain a solid sample. Washing solid sample with deionized water and anhydrous ethanol alternately for 6 times, vacuum drying the washed sample at 70 deg.C for 24 hr to obtain black solid powder as Fe0.8Ni0.2S2the/GO mimic enzyme contains 10% of graphene by mass.
(2) Product characterization
As shown in FIG. 1, Fe0.8Ni0.2S2The X-ray diffraction pattern of the/GO mimic enzyme has diffraction peaks at 2 theta values (29.9 degrees, 33.1 degrees, 37.2 degrees, 41.0 degrees, 47.3 degrees and 56.0 degrees) corresponding to [ Fe, Ni ] respectively]S2The (111), (200), (210), (211), (220) and (311) crystal faces of the card (PDF # 03-1131). No impurity peak was observed, indicating that the sampleThe purity is high. Measuring the contents of Fe and Ni in the mimic enzyme molecules by ICP (inductively coupled plasma) test to obtain Fe-Ni sulfide (Fe) in the mimic enzyme0.8Ni0.2S2。
As shown in FIG. 2, Fe0.8Ni0.2S2The transmission electron microscope image of the/GO mimic enzyme shows that the flaky iron-nickel sulfide is distributed on the graphene sheet.
As shown in FIG. 3, Fe0.8Ni0.2S2An EDX-mapping diagram of the/GO mimic enzyme shows that Fe, Ni and S elements exist, and iron-nickel sulfide is uniformly dispersed on the surface of graphene.
As shown in FIG. 4, Fe0.8Ni0.2S2The XPS patterns of the/GO mimetic enzymes showed the presence of Fe, Ni, S and C elements, as well as Fe and Ni with different oxidation numbers.
Example 2
Preparation of mimic enzyme: 3.636g Fe (NO) were weighed out3)3·9H2O、0.291g Ni(NO3)2·6H2O (molar ratio of iron to nickel is 9:1) is added into 40mL of deionized water, ultrasonic dissolution is carried out, and then 1.200g of Na is added2S·9H2And O, continuously carrying out ultrasonic dissolution and uniformly mixing. 0.060g of graphene was added to 10ml of deionized water to obtain a suspension, 2.5mg of sodium dodecylbenzenesulfonate was added and mixed well. The two prepared solutions are mixed and transferred into a 100mL polytetrafluoroethylene reaction kettle, the reaction temperature is 100 ℃, and the reaction time is 20 hours. And after the reaction is finished, naturally cooling the reacted mixture to room temperature, and performing centrifugal separation to obtain a solid sample. Washing solid sample with deionized water and anhydrous ethanol alternately for 6 times, vacuum drying the washed sample at 50 deg.C for 30 hr to obtain black solid powder as Fe0.8Ni0.2S2the/GO mimic enzyme contains 5% of graphene by mass.
Example 3
Preparation of mimic enzyme: 3.636g Fe (NO) were weighed out3)3·9H2O、0.291g Ni(NO3)2·6H2O (molar ratio of iron to nickel is 9:1) is added into 40mL of deionized water and dissolved by ultrasonic, and 4.800g of Na is added2S·9H2And O, continuously carrying out ultrasonic dissolution and uniformly mixing. 0.300g of graphene is added into 10ml of deionized water to obtain a suspension, 16.0mg of sodium dodecyl benzene sulfonate is added, and the mixture is uniformly mixed. The two prepared solutions are mixed and transferred into a 100mL polytetrafluoroethylene reaction kettle, the reaction temperature is 150 ℃, and the reaction time is 10 hours. And after the reaction is finished, naturally cooling the reacted mixture to room temperature, and performing centrifugal separation to obtain a solid sample. Washing solid sample with deionized water and anhydrous ethanol alternately for 6 times, vacuum drying the washed sample at 80 deg.C for 20 hr to obtain black solid powder as Fe0.8Ni0.2S2the/GO mimic enzyme contains 15% of graphene by mass.
Peroxidase-like activity detection
Respectively preparing 75 mu L of Fe with the concentration of 2mg/mL and the graphene mass fraction of 10% at the temperature of 25 DEG C0.8Ni0.2S2Aqueous solution of/GO mimic enzyme and Fe containing graphene with mass fraction of 15%0.8Ni0.2S2Aqueous solution of/GO mimic enzyme and Fe containing 5% of graphene by mass fraction0.8Ni0.2S2Aqueous solution of/GO mimic enzyme, aqueous solution of graphene and Fe0.8Ni0.2S2An aqueous solution. The aqueous solutions prepared above were added to 2.775mL of HAc-NaAc (pH 4.0,0.2M) buffer, and 75 μ L of deionized water was added to the blank control. To the above solution were further added 75. mu.L of 40mM 3,3 ', 5, 5' -Tetramethylbenzidine (TMB) and 75. mu.L of 10mM hydrogen peroxide to obtain a mixed solution. The resulting mixed solution was incubated in a thermostatic water bath at 25 ℃ for 1.0min, and the solution changed from colorless to blue. The characteristic peak of TMB at 652nm was detected by UV-visible spectrophotometer and the result is shown in FIG. 5. Visible and Fe0.8Ni0.2S2Compared with the prior art, the activity of the mimic enzyme is obviously improved by adding the graphene.
Effect of pH and temperature on the Activity of the mimic enzyme
(1) To 2.775mL of HAc-NaAc (pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 0.2M) buffer was added 75 μ L of Fe containing 10% graphene mass fraction at 2mg/mL at 25 ℃0.8Ni0.2S2The enzyme solution was simulated by GO, and then 75. mu.L of TMB with a concentration of 40mM and 75. mu.L of hydrogen peroxide with a concentration of 10mM were added, respectively, and incubated in a water bath at 25 ℃ for 1min, and then the absorbance at 652nm was measured, and the test results are shown in FIG. 6 (a). It can be seen that the absorbance change of the solution is large within the pH value range of 3.0-6.0, and the activity of the mimic enzyme is highest between the pH value of 3.0-4.0.
(2) In the above experiment, a buffer solution with pH 4.0 was selected, 10 parts of the same mixture was prepared without changing the other components, the mixture was incubated in a water bath at 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 ℃ for 1min, and the absorbance at 652nm was measured, and the test results are shown in fig. 6 (b). It can be seen that the activity of the mimic enzyme is enhanced with the increase of temperature, and the mimic enzyme has a wider applicable temperature range.
Detection of hydrogen peroxide
(1) 75 mu L of Fe with the concentration of 2mg/mL and the graphene mass fraction of 10%0.8Ni0.2S2Aqueous solution of/GO mimic enzyme, 75 μ L of TMB at 40mM concentration and 75 μ L of H at different concentrations2O2The resulting mixture was sequentially added to 2.775mL of HAc-NaAc buffer solution (pH 4.0,0.2M) to obtain mixed solutions having concentrations of hydrogen peroxide of 0.50, 1.25, 2.50, 6.25, 12.50, 18.75, 25.00, 37.50, 50.00, 62.50, and 125.00 μ M, respectively. The mixture was incubated in a water bath at 25 ℃ for 1min, and its absorbance at 652nm was measured, as shown in FIG. 7A.
(2) On the basis of the experiment (1), the concentration of other components is unchanged, and the concentration of hydrogen peroxide is changed to prepare mixed solution with the concentration of 0.50, 1.25, 2.50, 3.75, 5.00, 6.25, 7.50, 10.00 and 12.50 mu M of hydrogen peroxide. The mixture was incubated in a water bath at 25 ℃ for 1min, and its absorbance at 652nm was measured, as shown in FIG. 7B. The absorbance and the concentration of hydrogen peroxide have good linear relation and correlation coefficient R20.998. The LOD of the mimic enzyme to the hydrogen peroxide is calculated to be 0.29 mu M by using a formula detection limit LOD which is 3 sigma/k (sigma is the stability of a testing instrument, and k is the slope of the absorbance and the hydrogen peroxide straight line), and compared with the reference documents in the table 1, Fe0.8Ni0.2S2The detection time of the/GO mimic enzyme is shortest, and the LOD value is smallest.
Table 1: peroxidase assay H2O2Comparison of
Detection of Ascorbic Acid (AA)
(1) 75 mu L of Fe with the concentration of 2mg/mL and the graphene mass fraction of 10%0.8Ni0.2S2Aqueous solution of GO mimic enzyme, 75 μ L of TMB 10 μ L of AA with concentration of 40mM and 75 μ L of H with concentration of 10mM2O2Then, the solution was sequentially added to 2.765mL of HAc-NaAc buffer solution (pH 4.0,0.2M) to obtain mixed solutions having AA concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, and 140 μ M, respectively. The mixture was incubated in a water bath at 25 ℃ for 1min, and its absorbance at 652nm was measured, as shown in FIG. 8A. The absorbance of the solution at 625nm decreases with increasing AA concentration, and approaches 0 at an AA concentration of 140. mu.M.
(2) On the basis of the experiment (1), mixed solutions with AA concentrations of 3.33333, 10.00000, 16.66667, 26.66667, 33.33333, 43.33333 and 50.00000 μ M were prepared by changing the concentration of AA without changing other components. The mixture was incubated in a water bath at 25 ℃ for 1min, and its absorbance at 652nm was measured, as shown in FIG. 7B. The absorbance difference and the AA concentration have good linear relation, and the correlation coefficient R2When the result was 0.988, Fe0.8Ni0.2S2the/GO mimic enzyme can be used as a mimic enzyme to detect AA. At 25 deg.C, Fe0.8Ni0.2S2The detection time of the/GO mimic enzyme is 1.0min, and is the shortest compared with the existing reference (Table 2).
Table 2: comparison of peroxidase detection of AA
(3) Diluting the purchased juice by 100 times with deionized water, extracting strawberry and small tomato by a juicer to obtain extract, adding deionized water to prepare diluted solution with 100 times dilution, taking 10 μ L of diluted sample solution, and adding 10 μ L of standard ascorbic acid solution with concentration of 5, 15, 25 μ M respectively. 75 mu L of mimic enzyme aqueous solution with the concentration of 2mg/mL and the graphene mass fraction of 10%, 75 mu L of mixed solution of 40mM TMB, 20 mu L of sample dilute solution and AA, and 75 mu L of H with the concentration of 10mM2O2To 2.755mL of HAc-NaAc buffer solution (pH 4.0, 0.2M). The AA concentration in the sample is detected by adopting a standard addition method, and the test result is shown in Table 3, wherein the recovery rate is 98.1-105.6%, and the relative standard deviation is 0.17-1.88%. The results show that Fe0.8Ni0.2S2the/GO mimic enzyme is used for detecting AA and has high sensitivity.
TABLE 3 detection of AA in real samples
Claims (7)
1. An ascorbic acid mimic enzyme with high efficiency, wherein the mimic enzyme comprises Fe-Ni sulfide Fe0.8Ni0.2S2And graphene, iron nickel sulfide Fe0.8Ni0.2S2Uniformly dispersed on the graphene sheet layer.
2. The method for detecting ascorbate mimic enzyme according to claim 1, wherein said mimic enzyme is a lamellar two-dimensional structure.
3. The method for efficiently detecting the ascorbic acid mimic enzyme according to claim 1, wherein the mimic enzyme contains 5-15% of graphene by mass.
4. The method for preparing the high-efficiency detection ascorbic acid mimetic enzyme according to claim 1, comprising the steps of:
(1) mixing Fe (NO)3)3·9H2O、Ni(NO3)2·6H2O and Na2S·9H2Dissolving O in water, and adding a graphene aqueous solution containing sodium dodecyl benzene sulfonate to obtain a mixed solution;
(2) reacting the mixed solution at a constant temperature of 100-150 ℃ for 10-20 hours;
(3) and (3) cooling the reacted mixed material obtained in the step (2) to room temperature, performing centrifugal separation to obtain a solid product, alternately cleaning the solid product by using deionized water and ethanol, and finally performing vacuum drying at the temperature of 50-80 ℃ for 20-30 hours to obtain the mimic enzyme.
5. The method for preparing the enzyme mimic ascorbate with high efficiency according to claim 4, wherein the step (1) is Fe (NO)3)3·9H2O、Ni(NO3)2·6H2O and Na2S·9H2The molar ratio of O is 9:1: 5-20, and the mass of water is 6-7 times of the mass of solid.
6. The method for preparing the high-efficiency detection ascorbic acid mimic enzyme according to claim 4, wherein the mass ratio of the graphene, the sodium dodecyl benzene sulfonate and the water in the step (1) is 120-150: 5-8: 10000.
7. The method for preparing the enzyme mimic ascorbate with high efficiency according to claim 4, wherein the step (1) is Fe (NO)3)3·9H2The mass ratio of O to graphene is 303: 5 to 25.
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| CN108636425A (en) * | 2018-05-14 | 2018-10-12 | 潍坊学院 | Ferronickel sulfide-graphene composite material, preparation method and application |
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| CN108636425A (en) * | 2018-05-14 | 2018-10-12 | 潍坊学院 | Ferronickel sulfide-graphene composite material, preparation method and application |
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