CN114289020B - Preparation method of magnetic nanometer mimic enzyme, prepared mimic enzyme and application of mimic enzyme in detection of mercury ions - Google Patents
Preparation method of magnetic nanometer mimic enzyme, prepared mimic enzyme and application of mimic enzyme in detection of mercury ions Download PDFInfo
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- 230000003278 mimic effect Effects 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- -1 mercury ions Chemical class 0.000 title claims description 30
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- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 claims abstract description 19
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- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims abstract description 6
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- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- QVCGXRQVUIKNGS-UHFFFAOYSA-L cobalt(2+);dichloride;hydrate Chemical compound O.Cl[Co]Cl QVCGXRQVUIKNGS-UHFFFAOYSA-L 0.000 description 1
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- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
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- VITRLXDSBBVNCZ-UHFFFAOYSA-K trichloroiron;hydrate Chemical compound O.Cl[Fe](Cl)Cl VITRLXDSBBVNCZ-UHFFFAOYSA-K 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a preparation method of magnetic nanometer mimic enzyme, which relates to the technical field of mercury ion detection and 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 mol ratio of the cobalt chloride hexahydrate to the ferric trichloride hexahydrate to 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) Washing the reaction product in the step (2) under the attraction of the permanent magnet, and then drying. The invention also provides the magnetic nanometer mimic enzyme prepared by the method and application of the magnetic nanometer mimic enzyme in mercury ion detection. The invention has the beneficial effects that: the invention only adopts the precursor and the reducing agent, synthesizes the magnetic nanometer simulated enzyme by a simple hydrothermal method, and has mild reaction conditions; the strong magnetic property of the magnetic nanometer simulated enzyme is fully utilized, the washing process is simplified, and the synthesized magnetic nanometer 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 nanometer 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 the living standard of people, the importance of food safety is increasing. The mercury ions are used as heavy metal ions which are high in harm to human bodies and the environment, cannot be degraded by microorganisms, are easy to enrich in the human bodies, and are harmful to the human body health. Regulatory bodies such as the Environmental Protection Agency (EPA) and the World Health Organization (WHO) have regulated maximum allowable limits of 10nM and 30nM for mercury in drinking water, respectively, and it is therefore important to establish a method capable of rapidly detecting mercury ion content in water.
Currently, conventional 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, due to expensive and complex instrumentation, time consuming sample preparation and pre-concentration, these selective and sensitive techniques are limited to routine detection. Therefore, it becomes important how to establish a rapid, convenient, simple and reliable mercury ion detection method.
Nano-enzymes refer to nano-materials with catalytic activity, and since Fe 3O4 was found to have peroxidase activity in 2007, many nano-materials such as graphene, noble metals, and metal oxides were found to have enzyme-mimicking properties. 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 typically have peroxidase activity and are capable of catalyzing the oxidation of colorless TMB in the presence of hydrogen peroxide to produce blue oxTMB.
The patent with publication number CN103341360A discloses a nanomaterial mimic enzyme and application thereof in mercury ion detection, and after mercury ions are mixed with silver nanomaterial, various characteristic substrates of horseradish peroxidase can be catalytically oxidized in the presence of oxygen in the air, but the detection line is only 20nM.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of magnetic nanometer mimic enzyme capable of being used for detecting mercury ions, the prepared mimic enzyme and application thereof, wherein the detection line of the magnetic nanometer mimic enzyme is low.
The invention solves the technical problems by 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 mol ratio of the cobalt chloride hexahydrate to the ferric trichloride hexahydrate to 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) Washing the reaction product in the step (2) under the attraction of the permanent magnet, and then drying.
The beneficial effects are that: the invention only adopts the precursor and the reducing agent, synthesizes the magnetic nanometer simulated enzyme by a simple hydrothermal method, and has mild reaction conditions; the strong magnetic property of the magnetic nanometer simulated enzyme is fully utilized, the washing process is simplified, the synthesized magnetic nanometer material can be circularly and repeatedly used, and the detection line is 3nM when mercury ions are detected, and the detection line is low.
Preferably, the hydrothermal reaction temperature in the step (2) is 180-200 ℃ and the reaction time is 8-12h.
Preferably, in the step (3), washing with water and ethanol is performed three times each, and then vacuum drying is performed.
The magnetic nanometer mimic enzyme prepared by the method.
The beneficial effects are that: the magnetic nanometer mimic enzyme prepared by the invention can be used for detecting mercury ions, and the cysteine is used as a probe, so that the sensitivity is high, and the magnetic nanometer material can be recycled, so that the detection cost is reduced.
The application of the magnetic nanometer mimic enzyme prepared by the method in detecting mercury ions comprises the following steps:
(1) Adding magnetic nanometer mimic enzyme, hydrogen peroxide solution and 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 the volume ratio of 1:1 to obtain a complex solution;
(3) Mixing and incubating the mixed solution in the step (1) and the complexing solution in the step (2), measuring absorbance A at 652nm after the reaction is finished, using deionized water to replace mercury ion solution in a control group, measuring absorbance A 0 at 652nm after the reaction, taking mercury ion concentration as an abscissa, and taking delta A=A-A 0 as an ordinate, establishing a standard curve, and measuring the concentration of an unknown sample according to the standard curve.
The beneficial effects are that: the invention establishes a colorimetric sensing system for rapidly detecting Hg 2+ by utilizing the characteristic that cysteine can influence a CoFe 2O4-H2O2 -TMB system and Hg 2+ is specifically combined with the cysteine.
The invention can carry out semi-quantitative detection on Hg 2+ concentration by adopting a visual colorimetry, and can accurately and quantitatively detect Hg 2+ concentration by utilizing the absorbance of a UV-Vis measuring reaction system.
Preferably, the buffer solution is HAc-NaAc buffer, the pH value of the buffer solution is 3-6, the concentration of TMB solution is 0.1-0.9mM, and the concentration of hydrogen peroxide solution is 6-18mM.
Preferably, the reaction temperature in the step (1) is 20-40 ℃ and the reaction time is 30min.
Preferably, after the assay is completed, the magnetic nanomatrix is recovered by an externally applied magnetic field.
Preferably, the step (2) is incubated for 30min at room temperature.
Preferably, the step (3) is incubated for 30min at room temperature.
The invention has the advantages that: the invention only adopts the precursor and the reducing agent, synthesizes the magnetic nanometer simulated enzyme by a simple hydrothermal method, and has mild reaction conditions; the strong magnetic property of the magnetic nanometer simulated enzyme is fully utilized, the washing process is simplified, the synthesized magnetic nanometer material can be circularly and repeatedly used, and the detection line is 3nM when mercury ions are detected, and the detection line is low.
The magnetic nanometer mimic enzyme prepared by the invention can be used for detecting mercury ions, and the cysteine is used as a probe, so that the sensitivity is high, and the magnetic nanometer material can be recycled, so that the detection cost is reduced.
The invention establishes a colorimetric sensing system for rapidly detecting Hg 2+ by utilizing the characteristic that cysteine can influence a CoFe 2O4-H2O2 -TMB system and Hg 2+ is specifically combined with the cysteine.
The invention can carry out semi-quantitative detection on Hg 2+ concentration by adopting a visual colorimetry, and can accurately and quantitatively detect Hg 2+ concentration by utilizing the absorbance of a UV-Vis measuring reaction system.
Drawings
FIG. 1 is a schematic diagram of Hg 2+ detection by nanoenzyme activity in an embodiment of the present invention;
FIG. 2 is a graph of the ultraviolet absorption spectrum of the catalytic activity of the cobalt ferrite nanomaterial peroxide-mimetic enzyme in the presence and absence of Hg 2+ in example 7 of the present invention;
FIG. 3 is a graph showing the color change of different concentrations of mercury ions in the reaction system according to example 7 of the present invention;
FIG. 4 is a graph showing the linear relationship between the detection of Hg 2+ at different concentrations in example 7 of the present invention;
FIG. 5 is a graph showing the calibration of example 7 of the present invention;
FIG. 6 is an electron microscope image of the magnetic nano-enzyme according to example 7 of the present invention;
FIG. 7 is an electron microscope image of the magnetic nanoenzyme of example 7 of the invention in combination with the substrate TMB;
FIG. 8 is a graph showing the effect of pH of HAc-NaAc buffer solution on catalytic effect in examples 7, 8-13 of the present invention;
FIG. 9 is a graph showing the effect of incubation temperature on catalytic effect in examples 7, 14-17 of the present invention;
FIG. 10 is a graph showing the effect of TMB solution on catalytic effect in examples 7, 18-22 of the present invention;
FIG. 11 is a graph showing the effect of H 2O2 solution on the catalytic effect in examples 7, 23-27 of the present invention;
FIG. 12 is a graph showing the recycling efficiency of the magnetic cobalt ferrite nano-enzyme in example 7 of the present invention;
FIG. 13 is a graph showing the results of the selective detection of magnetic cobalt ferrite nanoenzymes for different metal ions in examples 7 and 31-39 according to the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. 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 be within the scope of the invention.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
Example 1
The preparation method of the magnetic nanometer mimic enzyme specifically comprises the following steps:
(1) 2mmol of cobalt chloride hydrate (CoCl 2·6H2 O), 4mmol of ferric chloride hydrate (FeCl 3·6H2 O) and 16mmol of sodium hydroxide are dissolved in 30mL of ethylene glycol, stirred and dissolved, and then ultrasonic treatment is carried out 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 black suspension;
(3) And (3) under the action of the 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 nanometer simulated enzyme.
Example 2-example 6
Examples 2 to 6 were different in the ratio of the respective raw materials, the reaction temperature and the reaction time, and the specific differences are shown in Table 1.
Table 1 shows the amounts of the raw materials and the reaction conditions in examples 2 to 6
Example 7
The magnetic nano-mimic enzyme in example 2 is used for detecting mercury ions (Hg 2+), the detection schematic diagram is shown in FIG. 1, and the specific steps are as follows:
(1) Preparing 1mg/mL cobalt ferrite nano enzyme suspension (water as a solvent), preparing HAc-NaAc buffer solutions (0.1M) with different pH values, and preparing TMB solutions, H 2O2 solutions and mercury ion standard solutions with different concentrations.
(2) 100. Mu.L of 1mg/mL cobalt ferrite magnetic nanoparticles, 100. Mu.L of 0.7mM TMB aqueous solution, and 100. Mu.L of 16mM hydrogen peroxide aqueous solution were added to 400. Mu.L of HAc-NaAc (pH= 4.0,0.1M), and after 30 minutes of reaction, a blue oxTMB solution was obtained.
(3) 200. Mu.L of a 20. Mu.M aqueous solution of cysteine was mixed with 200. Mu.L of a mercury ion solution of different concentrations (10 -9-5*10-6 M) and incubated at 30℃for 30 minutes.
(4) The solutions from 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 absorbance A at 652 nm.
(5) The control group replaced the mercury ion solution with 200 μl deionized water and absorbance a 0 at 652nm was measured after the reaction. And (3) plotting by taking the mercury ion concentration as an abscissa and deltaA=A-A 0 as an ordinate, obtaining a mercury ion standard curve in a certain linear range, and measuring the concentration of an unknown sample according to the standard curve.
Fig. 2 is an ultraviolet absorption spectrum of the cobalt ferrite nanomaterial peroxide mimic enzyme catalytic activity in the presence and absence of Hg 2+, and it can be seen that the presence of mercury ions significantly inhibits the reaction of cobalt ferrite to catalyze TMB.
FIG. 3 is a graph showing the color change of different concentrations of mercury ions in a reaction system, wherein the concentration of mercury ions is from high to low and the color is from dark to light. As can be seen from the actual comparison picture, when the concentration of Hg 2+ is 10nM-5 mu M, the reaction system has obvious color change, so that the Hg 2+ concentration can be semi-quantitatively detected by visual change of the color of the reaction system.
Fig. 4 is a linear relation diagram of detection of different concentrations of Hg 2+, fig. 5 is a standard curve diagram, the colorimetric detection method has good reaction for detecting mercury ions, the detection range of Hg 2+ is 10-100nM, the detection limit is 3nM, the linear equation is y=0.98x+0.002, and the correlation coefficient R 2 =0.995.
Fig. 6 and 7 are respectively 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, and it can be seen that the magnetic nanoenzyme in the present embodiment has good dispersibility and high crystallinity, and can be combined with the substrate TMB with sufficient moisture.
Example 8
Examples 8-13 differ from example 7 in that: the pH of the HAc-NaAc buffer solution was adjusted to 3.0, 3.5, 4.5, 5.0, 5.5 and 6, respectively.
FIG. 8 is a graph showing the results of the catalytic conditions at different pH values, and it can be seen that the catalytic effect is optimal at a pH of 4.0 in example 7.
Example 14-example 17
Examples 14-17 differ from example 7 in that: the incubation temperatures in step (3) were adjusted to 20, 25, 35, 40 ℃ respectively.
FIG. 9 is a graph showing the results of the catalytic conditions at different temperatures, and it can be seen that the catalytic effect is optimal at a temperature of 30℃in example 7.
Example 18-example 22
Example 18-example 22 differs from example 7 in that: the incubation concentrations of TMB solutions were adjusted to 0.1, 0.3, 0.5, 0.8, 0.9mM, respectively.
FIG. 10 is a graph showing the results of the catalytic conditions at different TMB solution concentrations, and it can be seen that the catalytic effect is optimal at a TMB solution concentration of 0.7 mM.
Example 23-example 27
Examples 23-27 differ from example 7 in that: the concentrations of H 2O2 solutions were adjusted to 6, 10, 12, 14, 18mM, respectively.
FIG. 11 is a graph showing the results of the catalytic conditions at different concentrations of H 2O2 solution, and it can be seen that the catalytic effect is optimal when the concentration of H 2O2 solution is 16 mM.
Example 28
This embodiment differs from embodiment 7 in that: the volume of the cysteine aqueous 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 μl.
Example 30
The magnetic cobalt ferrite nanoenzymes in example 7, example 28 and example 29 were washed and dried again, reacted under the same conditions as before, and absorbance at 652nm was measured to verify the recycling efficiency.
Fig. 12 is a graph showing the recovery and recycling efficiency of the magnetic cobalt ferrite nano enzyme in example 7, and it can be seen that after 5 times of recycling, the simulated enzyme catalytic efficiency is still more than 80%, which proves that the method can be used as a method for detecting mercury ions with high circulation efficiency.
Examples 31 to 39
This embodiment differs from embodiment 7 in that: the mercury ion solution was replaced by Zn2+、Al3+、Cu2+、Na+、Ca2+、Mg2+、K+、Pb2+、Ni2+ solutions, respectively.
FIG. 13 is a graph of the results of selective detection of different metal ions, showing the superiority of the present invention for specific detection of mercury ions.
Comparative example 1
The magnetic nano-mimic enzyme in example 2 is used for detecting sulfite ions, and the detection method is the same as that of example 7 in the patent with publication number of CN 111203221A, and the cobalt ferrite nanocluster mimic enzyme is replaced by the same amount of the magnetic nano-mimic enzyme in example 2.
The detection limit is found to be 1 x 10 -8 M through detection, which is far lower than the detection limit of 5 x 10 -6 M in the CN 111203221A patent, and the detection limit is lower.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the 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 scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. The application of the magnetic nano mimic enzyme in detecting mercury ions is characterized in that: the method comprises the following steps:
(1) Adding magnetic nanometer mimic enzyme, hydrogen peroxide solution and 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 the volume ratio of 1:1 to obtain a complex solution;
(3) Mixing and incubating the mixed solution in the step (1) and the complexing solution in the step (2), measuring absorbance A at 652nm after the reaction is finished, using deionized water to replace mercury ion solution in a control group, measuring absorbance A0 at 652nm after the reaction, taking mercury ion concentration as an abscissa, and taking delta A=a-A0 as an ordinate, establishing a standard curve, and measuring the concentration of an unknown sample according to the standard curve;
The preparation method of the magnetic nanometer mimic enzyme comprises the following specific steps:
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 mol ratio of the cobalt chloride hexahydrate to the ferric trichloride hexahydrate to the sodium hydroxide is 1:2:16;
b, carrying out hydrothermal reaction on the solution in the step a, and cooling to room temperature after the reaction;
c washing the reaction product in the step b under the attraction of the permanent magnet, and then drying.
2. The use of the magnetic nano-mimic enzyme according to claim 1 for detecting mercury ions, wherein: the hydrothermal reaction temperature in the step b is 180-200 ℃ and the reaction time is 8-12h.
3. The use of the magnetic nano-mimic enzyme according to claim 1 for detecting mercury ions, wherein: the step c is washed three times with water and ethanol, and then vacuum-dried.
4. The use of the magnetic nano-mimic enzyme according to claim 1 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 TMB solution is 0.1-0.9mM, and the concentration of hydrogen peroxide solution is 6-18mM.
5. The use of the magnetic nano-mimic enzyme according to claim 1 for detecting mercury ions, wherein: the reaction temperature in the step (1) is 20-40 ℃ and the reaction time is 30min.
6. The use of the magnetic nano-mimic enzyme according to claim 1 for detecting mercury ions, wherein: after the measurement is completed, the magnetic nanometer simulated enzyme is recovered by an external magnetic field.
7. The use of the magnetic nano-mimic enzyme according to claim 1 for detecting mercury ions, wherein: the step (2) is incubated for 30min at room temperature.
8. The use of the magnetic nano-mimic enzyme according to claim 1 for detecting mercury ions, wherein: the step (3) is incubated for 30min at room temperature.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111564208.2A CN114289020B (en) | 2021-12-20 | 2021-12-20 | Preparation method of magnetic nanometer mimic enzyme, prepared mimic enzyme and application of mimic enzyme in detection of mercury ions |
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