CN114289020A - Preparation method of magnetic nano mimic enzyme, prepared mimic enzyme and application of mimic enzyme in detection of mercury ions - Google Patents
Preparation method of magnetic nano mimic enzyme, prepared mimic enzyme and application of mimic enzyme in detection of mercury ions Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
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- 229910052753 mercury Inorganic materials 0.000 title claims description 30
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 21
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Abstract
The invention discloses a preparation method of magnetic nano mimic enzyme, relating to the technical field of mercury ion detection and comprising the following steps: (1) dissolving cobalt chloride hexahydrate, ferric trichloride hexahydrate and sodium hydroxide in ethylene glycol, stirring and dissolving, and performing ultrasonic treatment to form a solution, wherein the molar ratio of the cobalt chloride hexahydrate, the ferric trichloride hexahydrate and the sodium hydroxide is 1:2: 16; (2) carrying out hydrothermal reaction on the solution in the step (1), and cooling to room temperature after the reaction; (3) and (3) washing the reaction product in the step (2) under the attraction of a permanent magnet, and then drying. The invention also provides the magnetic nano mimic enzyme prepared by the method and application of the magnetic nano mimic enzyme in mercury ion detection. The invention has the beneficial effects that: the invention only adopts a precursor and a reducing agent, synthesizes the magnetic nano mimic enzyme by a simple hydrothermal method, and has mild reaction conditions; the strong magnetic property of the magnetic nano mimic enzyme is fully utilized, the washing process is simplified, and the synthesized magnetic nano material can be recycled.
Description
Technical Field
The invention relates to the technical field of mercury ion detection, in particular to a preparation method of magnetic nano mimic enzyme, the prepared mimic enzyme and application of the mimic enzyme in detection of mercury ions.
Background
In recent years, with the improvement of living standard of people, the importance degree of food safety is continuously increased. As a heavy metal ion which is highly harmful to human bodies and the environment, the mercury ion not only can not be degraded by microorganisms, but also is easy to enrich in human bodies and is harmful to human health. Regulatory bodies such as the Environmental Protection Agency (EPA) and the World Health Organization (WHO) have specified maximum allowable limits for mercury in drinking water of 10nM and 30nM, respectively, and it has become important to establish a method capable of rapidly detecting the content of mercury ions in water.
At present, the traditional methods for detecting mercury ions mainly include atomic absorption spectrometry (CVAAS), atomic absorption/emission spectrometry (AAS/AES), inductively coupled plasma mass spectrometry (ICP-MS), atomic-fluorescence spectrometry (AFS), High Performance Liquid Chromatography (HPLC), Ion Selective Electrode (ISE), flame photometry and stripping voltammetry. However, these selective and sensitive techniques are limited to routine detection due to expensive and complex instruments, time consuming sample preparation and preconcentration. Therefore, how to establish a rapid, convenient, simple and reliable mercury ion detection method becomes important.
Nanoenzymes refer to nanomaterials with catalytic activity, since the discovery of Fe in 20073O4Since the peroxidase activity, many nanomaterials such as graphene, noble metals and metal oxides are found to have the property of mimic enzyme. Compared with natural enzymes, the nano-enzyme has the advantages of high catalytic activity, good stability, low price and the like, so that the nano-enzyme is widely applied to the aspects of analysis and detection, biomedicine and the like. Nanoenzymes generally have peroxidase activity and are capable of catalytically oxidizing colorless TMB in the presence of hydrogen peroxide to produce a blue oxTMB.
Patent publication No. CN103341360A discloses a nanomaterial mimic enzyme and its application in mercury ion detection, wherein mercury ions and silver nanomaterials can be mixed to catalytically oxidize various characteristic substrates of horseradish peroxidase in the presence of oxygen in the air, but the detection line is only 20 nM.
Disclosure of Invention
The invention aims to provide a preparation method of a magnetic nano mimic enzyme capable of being used for detecting mercury ions, the prepared mimic enzyme and application thereof.
The invention solves the technical problems through the following technical means:
the preparation method of the magnetic nanometer mimic enzyme comprises the following steps:
(1) dissolving cobalt chloride hexahydrate, ferric trichloride hexahydrate and sodium hydroxide in ethylene glycol, stirring and dissolving, and performing ultrasonic treatment to form a solution, wherein the molar ratio of the cobalt chloride hexahydrate, the ferric trichloride hexahydrate and the sodium hydroxide is 1:2: 16;
(2) carrying out hydrothermal reaction on the solution in the step (1), and cooling to room temperature after the reaction;
(3) and (3) washing the reaction product in the step (2) under the attraction of a permanent magnet, and then drying.
Has the advantages that: the invention only adopts a precursor and a reducing agent, synthesizes the magnetic nano mimic enzyme by a simple hydrothermal method, and has mild reaction conditions; the strong magnetic property of the magnetic nano mimic enzyme is fully utilized, the washing process is simplified, the synthesized magnetic nano material can be recycled, the detection line is 3nM when the mercury ions are detected, and the detection line is low.
Preferably, the hydrothermal reaction temperature in the step (2) is 180-.
Preferably, the step (3) is washed with water and ethanol three times each, and then vacuum-dried.
The magnetic nanometer mimic enzyme prepared by the method is adopted.
Has the advantages that: the magnetic nano mimic enzyme prepared by the invention can be used for detecting mercury ions, cysteine is used as a probe, the sensitivity is high, and the magnetic nano material can be recycled, so that the detection cost is reduced.
The application of the magnetic nano mimic enzyme prepared by the method in detecting mercury ions comprises the following steps:
(1) adding the magnetic nano mimic enzyme, a hydrogen peroxide solution and a TMB solution into a buffer solution for reaction to obtain a mixed solution;
(2) mixing and incubating cysteine solution and mercury ion solutions with different concentrations according to a volume ratio of 1:1 to obtain a complex solution;
(3) mixing and incubating the mixed solution obtained in the step (1) and the complexing solution obtained in the step (2), measuring the absorbance A at 652nm after the reaction is finished, replacing the mercury ion solution with deionized water for a control group, and measuring the absorbance A at 652nm after the reaction0On the abscissa, the concentration of mercury ions is defined as Δ a ═ a-a0And establishing a standard curve for the ordinate, and measuring the concentration of the unknown sample according to the standard curve.
Has the advantages that: the invention can affect CoFe by using cysteine2O4-H2O2-TMB system, and Hg2+Has the characteristic of specific combination with cysteine, establishes a rapid detection method for Hg2+The colorimetric sensing system of (1).
The present invention can use visual colorimetry to remove Hg2+The concentration is semi-quantitatively detected, and the absorbance of a reaction system can be measured by using UV-Vis to accurately and quantitatively detect Hg2+The concentration of (c).
Preferably, the buffer solution is HAc-NaAc buffer, the pH value of the buffer solution is 3-6, the concentration of the TMB solution is 0.1-0.9mM, and the concentration of the hydrogen peroxide solution is 6-18 mM.
Preferably, the reaction temperature in the step (1) is 20-40 ℃, and the reaction time is 30 min.
Preferably, after the determination is completed, the magnetic nano mimic enzyme is recovered by applying a magnetic field.
Preferably, said step (2) is incubated at room temperature for 30 min.
Preferably, said step (3) is incubated at room temperature for 30 min.
The invention has the advantages that: the invention only adopts a precursor and a reducing agent, synthesizes the magnetic nano mimic enzyme by a simple hydrothermal method, and has mild reaction conditions; the strong magnetic property of the magnetic nano mimic enzyme is fully utilized, the washing process is simplified, the synthesized magnetic nano material can be recycled, the detection line is 3nM when the mercury ions are detected, and the detection line is low.
The magnetic nano mimic enzyme prepared by the invention can be used for detecting mercury ions, cysteine is used as a probe, the sensitivity is high, and the magnetic nano material can be recycled, so that the detection cost is reduced.
The invention can affect CoFe by using cysteine2O4-H2O2-TMB system, and Hg2+Has the characteristic of specific combination with cysteine, establishes a rapid detection method for Hg2+The colorimetric sensing system of (1).
The present invention can use visual colorimetry to remove Hg2+The concentration is semi-quantitatively detected, and the absorbance of a reaction system can be measured by using UV-Vis to accurately and quantitatively detect Hg2+The concentration of (c).
Drawings
FIG. 1 shows the detection of Hg by nanoenzyme activity in the present invention2+A schematic diagram;
FIG. 2 shows Hg in example 7 of the present invention2+Ultraviolet absorption spectrograms of the catalytic activity of the cobalt ferrite nanomaterial peroxidase simulant in the presence and absence;
FIG. 3 is a graph showing the color change of mercury ions in different concentrations in the reaction system in example 7 of the present invention;
FIG. 4 shows different Hg concentrations in example 7 of the present invention2+A detected linear relation graph;
FIG. 5 is a standard curve chart in example 7 of the present invention;
FIG. 6 is an electron micrograph of a magnetic nanoenzyme in example 7 of the present invention;
FIG. 7 is an electron micrograph of a magnetic nanoenzyme bonded to a substrate TMB in example 7 of the present invention;
FIG. 8 is a graph showing the effect of the pH value of the HAc-NaAc buffer solution on the catalytic effect in examples 7, 8 to 13 of the present invention;
FIG. 9 is a graph showing the effect of incubation temperature on catalytic performance in examples 7 and 14 to 17;
FIG. 10 is a graph showing the effect of TMB solution on the catalytic effect in examples 7, 18 to 22;
FIG. 11 shows H in examples 7, 23 to 27 of the present invention2O2A result graph of the influence of the solution on the catalytic effect;
FIG. 12 is a graph showing the recycling efficiency of magnetic cobalt ferrite nanoenzyme in example 7 of the present invention;
FIG. 13 is a diagram showing the results of selective detection of different metal ions by the magnetic cobalt ferrite nanoenzyme in examples 7, 31 to 39 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The specific techniques or conditions not specified in the examples can be performed according to the techniques or conditions described in the literature in the field or according to the product specification.
Example 1
The preparation method of the magnetic nano mimic enzyme specifically comprises the following steps:
(1) 2mmol of cobalt chloride hydrate (CoCl)2·6H2O), 4mmol of hydrated ferric chloride (FeCl)3·6H2O) and 16mmol of sodium hydroxide are dissolved in 30mL of glycol, and after stirring and dissolving, the solution is subjected to ultrasonic treatment for 30 minutes to form a yellow solution;
(2) putting the solution obtained in the step (1) into a polytetrafluoroethylene container, putting the polytetrafluoroethylene container into an oven, reacting for 8 hours at 180 ℃, naturally cooling to room temperature, and taking out a black suspension;
(3) and (3) under the action of a permanent magnet, washing the suspension in the step (2) with deionized water and ethanol for three times respectively, and drying in vacuum to obtain the magnetic nano mimic enzyme.
Example 2 to example 6
The differences between examples 2 and 6 are that the raw material ratios, the reaction temperatures and the reaction times are different, and the specific differences are shown in table 1.
Table 1 shows the amounts of the respective raw materials and the reaction conditions in examples 2 to 6
Example 7
Detection of mercury ions (Hg) Using the magnetic Nanometric mimetic enzyme of example 22+) The detection schematic diagram is shown in fig. 1, and the specific steps are as follows:
(1) preparing 1mg/mL cobalt ferrite nanoenzyme suspension (water as solvent), HAc-NaAc buffer solutions (0.1M) with different pH values, and TMB solution and H solution with different concentrations2O2Solution and mercury ion standard solution.
(2) mu.L of 1mg/mL cobalt ferrite magnetic nanoparticles, 100. mu.L of a 0.7mM aqueous TMB solution, and 100. mu.L of a 16mM aqueous hydrogen peroxide solution were added to 400. mu.L HAc-NaAc (pH 4.0, 0.1M), and a blue oxTMB solution was obtained after a reaction for 30 minutes.
(3) 200. mu.L of 20. mu.M aqueous cysteine solution was mixed with 200. mu.L of a solution having a concentration different from that of 200. mu.L (10)-9-5*10-6M) and incubated at 30 ℃ for 30 minutes.
(4) The solutions in steps (2) and (3) were mixed and incubated at room temperature for 10 minutes with a clear color change, and the resulting final reaction solution was measured for its absorbance value A at 652 nm.
(5) In the control group, 200. mu.L of deionized water was used in place of the mercury ion solution, and the absorbance A at 652nm was measured after the reaction0. Taking the concentration of mercury ions as the abscissa, Delta A is A-A0And drawing a vertical coordinate, obtaining a mercury ion standard curve in a certain linear range, and measuring the concentration of the unknown sample according to the standard curve.
FIG. 2 is Hg2+In the presence and absence of the cobalt ferrite nano material, the peroxide simulates the ultraviolet absorption spectrum of the catalytic activity of the enzyme, and the existence of mercury ions can obviously inhibit the reaction of catalyzing TMB by the cobalt ferrite.
FIG. 3 is a diagram showing the color change of mercury ions of different concentrations in a reaction system, wherein the concentration of the mercury ions is changed from high to low, and the color is changed from dark to light. As can be seen from the actual comparative picture, when Hg is present2+Since the reaction system has a significant color change at a concentration of 10nM to 5. mu.M, the reaction system can be visualized for Hg by visual observation of the color change2+And (5) carrying out semi-quantitative detection on the concentration.
FIG. 4 shows different Hg concentrations2+Linear relationship diagram of detection, fig. 5 is a standard curve diagram, the colorimetric detection method has good reaction for detecting mercury ions, Hg2+The detection range of (1) is 10-100nM, the detection limit is 3nM, the linear equation is Y-0.98X +0.002, and the correlation coefficient R2＝0.995。
Fig. 6 and 7 are an electron microscope image of the magnetic nanoenzyme and an electron microscope image of the magnetic nanoenzyme when the magnetic nanoenzyme is combined with the substrate TMB, respectively, and it can be seen that the magnetic nanoenzyme in this example has good dispersibility and high crystallinity, and can be combined with the substrate TMB with sufficient moisture.
Example 8
Example 8-example 13 differs from example 7 in that: the HAc-NaAc buffer solution was adjusted to pH 3.0, 3.5, 4.5, 5.0, 5.5 and 6, respectively.
FIG. 8 is a graph showing the results of catalytic conditions at different pH values, and it can be seen that the catalytic effect is the best in example 7 at pH 4.0.
Example 14-example 17
Example 14-example 17 differs from example 7 in that: the incubation temperatures in step (3) were adjusted to 20, 25, 35, and 40 ℃.
FIG. 9 is a graph showing the results of catalytic conditions at different temperatures, and it can be seen that the catalytic effect is the best in example 7 at a temperature of 30 ℃.
Example 18 example 22
Example 18-example 22 differs from example 7 in that: the incubation concentrations of the TMB solution were adjusted to 0.1, 0.3, 0.5, 0.8 and 0.9mM, respectively.
FIG. 10 is a graph showing the results of catalytic conditions at different concentrations of TMB solution, and it can be seen that the catalytic effect is the best when the concentration of TMB solution is 0.7 mM.
Example 23 example 27
Example 23-example 27 differs from example 7 in that: adjusting H2O2The concentrations of the solutions were 6, 10, 12, 14, 18mM, respectively.
FIG. 11 shows a case of a variation H2O2The result of the catalytic conditions at the solution concentration is shown in the figure, and H can be seen2O2The best catalytic effect is achieved when the concentration of the solution is 16 mM.
Example 28
This embodiment differs from embodiment 7 in that: the volume of the aqueous cysteine solution was adjusted to 100. mu.L.
Example 29
This embodiment differs from embodiment 7 in that: the volume of the mercury ion solution was adjusted to 100. mu.L.
Example 30
The magnetic cobalt ferrite nanoenzymes in example 7, example 28 and example 29 were washed and dried again, and the reaction was carried out under the same conditions as before, and the absorbance at 652nm was measured to verify the high efficiency of recycling.
FIG. 12 is a graph showing the recycling efficiency of the magnetic cobalt ferrite nanoenzyme of example 7, and it can be seen that the simulated enzyme catalytic efficiency remains above 80% after 5 cycles of recycling, demonstrating that the method can be used as a cyclic and efficient method for detecting mercury ions.
Example 31 example 39
This embodiment differs from embodiment 7 in that: respectively replacing the mercury ion solution with Zn2+、Al3+、Cu2+、Na+、Ca2+、Mg2+、K+、Pb2+、Ni2+And (3) solution.
FIG. 13 is a graph showing the results of selective detection of different metal ions, which shows the superiority of the present invention for specifically detecting mercury ions.
Comparative example 1
The magnetic nano mimic enzyme in the embodiment 2 is adopted to detect sulfite ions, the detection method is the same as that in the embodiment 7 in the patent with the publication number of CN 111203221A, and the cobalt ferrite nanocluster mimic enzyme is equivalently replaced by the magnetic nano mimic enzyme in the embodiment 2 of the invention.
The detection limit is 1 x 10 after detection-8M, far below the limit of detection of CN 111203221A patent by 5 x 10-6M, its detection limit is lower.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. The preparation method of the magnetic nano mimic enzyme is characterized by comprising the following steps: the method comprises the following steps:
(1) dissolving cobalt chloride hexahydrate, ferric trichloride hexahydrate and sodium hydroxide in ethylene glycol, stirring and dissolving, and performing ultrasonic treatment to form a solution, wherein the molar ratio of the cobalt chloride hexahydrate, the ferric trichloride hexahydrate and the sodium hydroxide is 1:2: 16;
(2) carrying out hydrothermal reaction on the solution in the step (1), and cooling to room temperature after the reaction;
(3) and (3) washing the reaction product in the step (2) under the attraction of a permanent magnet, and then drying.
2. The method for preparing magnetic nano mimic enzyme according to claim 1, wherein the method comprises the following steps: the hydrothermal reaction temperature in the step (2) is 180-200 ℃, and the reaction time is 8-12 h.
3. The method for preparing magnetic nano mimic enzyme according to claim 1, wherein the method comprises the following steps: and (4) washing the obtained product with water and ethanol for three times respectively in the step (3), and then carrying out vacuum drying.
4. The magnetic nano mimic enzyme prepared by the preparation method of any one of claims 1 to 3.
5. The application of the magnetic nano mimic enzyme prepared by the preparation method of any one of claims 1 to 3 in detection of mercury ions is characterized in that: the method comprises the following steps:
(1) adding the magnetic nano mimic enzyme, a hydrogen peroxide solution and a TMB solution into a buffer solution for reaction to obtain a mixed solution;
(2) mixing and incubating cysteine solution and mercury ion solutions with different concentrations according to a volume ratio of 1:1 to obtain a complex solution;
(3) mixing and incubating the mixed solution obtained in the step (1) and the complexing solution obtained in the step (2), measuring the absorbance A at 652nm after the reaction is finished, replacing the mercury ion solution with deionized water for a control group, and measuring the absorbance A at 652nm after the reaction0On the abscissa, the concentration of mercury ions is defined as Δ a ═ a-a0And establishing a standard curve for the ordinate, and measuring the concentration of the unknown sample according to the standard curve.
6. The use of the magnetic nanomanidase according to claim 5 for detecting mercury ions, wherein: the buffer solution is HAc-NaAc buffer, the pH value of the buffer solution is 3-6, the concentration of the TMB solution is 0.1-0.9mM, and the concentration of the hydrogen peroxide solution is 6-18 mM.
7. The use of the magnetic nanomanidase according to claim 5 for detecting mercury ions, wherein: the reaction temperature in the step (1) is 20-40 ℃, and the reaction time is 30 min.
8. The use of the magnetic nanomanidase according to claim 5 for detecting mercury ions, wherein: and after the determination is finished, the magnetic nano mimic enzyme is recovered by an external magnetic field.
9. The use of the magnetic nanomanidase according to claim 5 for detecting mercury ions, wherein: and (3) incubating at room temperature for 30 min.
10. The use of the magnetic nanomanidase according to claim 5 for detecting mercury ions, wherein: and (3) incubating at room temperature for 30 min.
Priority Applications (1)
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| CN115282966A (en) * | 2022-07-21 | 2022-11-04 | 河北科技师范学院 | Ferrous ion doped copper cobaltate material and preparation method thereof |
| CN116351382A (en) * | 2023-05-04 | 2023-06-30 | 四川农业大学 | Copper oxide and ferric oxide nano enzyme and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115121251A (en) * | 2022-07-11 | 2022-09-30 | 徐州医科大学 | Preparation method and application of magnetic FeCo bimetallic carbon-based porous nanoenzyme |
| CN115121251B (en) * | 2022-07-11 | 2024-04-23 | 徐州医科大学 | Preparation method and application of magnetic FeCo bimetallic carbon-based porous nano-enzyme |
| CN115282966A (en) * | 2022-07-21 | 2022-11-04 | 河北科技师范学院 | Ferrous ion doped copper cobaltate material and preparation method thereof |
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| CN116351382A (en) * | 2023-05-04 | 2023-06-30 | 四川农业大学 | Copper oxide and ferric oxide nano enzyme and preparation method and application thereof |
| CN116351382B (en) * | 2023-05-04 | 2024-04-09 | 四川农业大学 | Copper oxide and ferric oxide nano enzyme and preparation method and application thereof |
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