CN114272862B - Ruthenium-based metal ion liquid polymer microsphere artificial enzyme and preparation method and application thereof - Google Patents
Ruthenium-based metal ion liquid polymer microsphere artificial enzyme and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a ruthenium-based metal ion liquid polymer microsphere artificial enzyme, and a preparation method and application thereof. The preparation method comprises the following steps: (1) mixing a salicylic acid solution, a vinyl imidazole solution and a ruthenium trichloride solution, stirring, extracting, evaporating the obtained oil phase solution to remove a solvent, and drying to obtain ruthenium-based salicylic acid vinyl imidazole ionic liquid; (2) dispersing the ionic liquid obtained in the step (1) in a solvent, adding divinylbenzene for mixing, adding a free radical initiator for reaction, separating precipitate, washing with water, and drying to obtain solid powder; (3) and (3) dispersing the solid powder obtained in the step (2) in a solvent to obtain a reactant, adding a reducing agent for reaction, and drying the solid obtained by filtering to obtain the ruthenium-based metal ion liquid polymer microsphere artificial enzyme. The artificial enzyme prepared by the invention has good dispersibility and high oxidase-like activity, and can be used for detecting prochloraz by a colorimetric method.
Description
Technical Field
The invention relates to the field of preparation and application of artificial enzymes, in particular to a ruthenium-based metal ion liquid polymer microsphere artificial enzyme and a preparation method and application thereof.
Background
Strawberry fruits are bright red and beautiful, soft and juicy, sweet and sour, fragrant and rich in nutritive value, are known as 'fruit queen', and contain rich nutritive substances such as vitamin C, vitamin A, vitamin E, vitamin PP, vitamin B1, vitamin B2, carotene, tannic acid, aspartic acid, copper, strawberry amine, pectin, cellulose, folic acid, iron, calcium, ellagic acid, anthocyanin and the like. The strawberries are native to south America, China is introduced in the beginning of the 20 th century, the planting area is continuously enlarged in recent years, the strawberries become one of the largest strawberry production countries in the world, and the planting area keeps continuously growing market conditions. However, strawberries are very susceptible to diseases and insect pests due to their own growth characteristics. In addition, strawberries have a large water content and are susceptible to decay and deterioration caused by microorganisms after being picked, so that pesticides are widely used in the planting and storage processes of strawberries in order to improve the yield and appearance quality of strawberries [4 ]. The fruit and vegetable pesticide residue ranking list published by the U.S. environmental protection organization EWG in 2017 shows that 70% of fruit and vegetable samples have pesticide residues detected, wherein the strawberry pesticide residue is the highest.
The world health organization indicates that pesticides can be potentially toxic to humans, affect the reproductive system, immune system or nervous system of humans, and cause cancer and other problems. Prochloraz is a spectral bactericide, is usually used in the strawberry storage process, has obvious prevention effect on diseases caused by ascomycetes and deuteromycetes, is usually used in the strawberry storage process, and is easy to remain in strawberries. Therefore, the development of the accurate, rapid and high-sensitivity prochloraz residue detection method has important practical significance.
The main methods for detecting pesticide residues are chromatography, surface enhanced Raman scattering, fluorescence, electrochemistry and colorimetry. Chromatography is a common pesticide detection tool in laboratories and has high sensitivity and selectivity, but it requires tedious sample pretreatment, use of expensive instruments and skilled operators, greatly limiting its application in rapid field detection. The surface enhanced Raman scattering method has higher sensitivity and selectivity, but the instability of detection signals limits the practical application of the method. The fluorescence method has the advantages of rapidness, high sensitivity and high selectivity, but the interference of background fluorescence of biomacromolecules can influence the accurate determination of low-concentration pesticides. Electrochemical analysis methods have high sensitivity and rapidity, but some electrochemically active molecules may interfere with the detection of trace pesticide residues. The colorimetric method has low cost, simple operation and visual reading, thereby being concerned more. In the existing colorimetric method, natural oxidase is adopted. Since natural oxidase is easily inactivated, sensitive to temperature and pH, and difficult to store for a long time, it brings inconvenience to its practical application. Compared with natural enzyme, the metal nano-material artificial enzyme has higher stability and lower cost. However, the activity of the existing artificial enzyme is obviously lower than that of the natural enzyme.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a ruthenium-based metal ion liquid polymer microsphere artificial enzyme and a preparation method and application thereof. The ruthenium-based metal ion liquid polymer microsphere artificial enzyme is prepared by introducing ruthenium into a combination with a free radical initiator, can be used for detecting prochloraz by combining a colorimetric method, and has the advantages of low cost, simple operation, visual reading, no need of fussy sample pretreatment, short detection time (only 30 min) and obviously lower detection limit than that of the existing method.
The technical scheme of the invention is as follows:
a preparation method of an artificial enzyme of ruthenium-based metal ion liquid polymer microspheres comprises the following steps:
(1) mixing a salicylic acid solution, a vinyl imidazole solution and a ruthenium trichloride solution, stirring, extracting, evaporating the obtained oil phase solution to remove a solvent, and drying to obtain ruthenium-based salicylic acid vinyl imidazole ionic liquid;
(2) dispersing the ruthenium-based salicylic acid vinyl imidazole ionic liquid obtained in the step (1) in a solvent, adding divinylbenzene for mixing, adding a free radical initiator for reaction, separating precipitate, washing with water, and drying to obtain solid powder;
(3) and (3) dispersing the solid powder obtained in the step (2) in a solvent to obtain a reactant, adding a reducing agent for reaction, and drying the solid obtained by filtering to obtain the ruthenium-based metal ion liquid polymer microsphere artificial enzyme.
Further, in the step (1), the molar concentrations of the salicylic acid solution, the vinylimidazole solution and the ruthenium trichloride solution are all 0.05-1.2 mol/L; the volume ratio of the salicylic acid solution to the vinyl imidazole solution to the ruthenium trichloride solution is 6: 5-6: 0.5-1.5.
Further, in the step (1), the stirring speed is 60-80r/min, and the time is 45-60 min; the extracting agent used for extraction is dichloromethane; the evaporation is rotary evaporation; the drying temperature is 70-100 ℃, and the drying time is 8-14 h.
Further, in the step (2), the solvent is absolute ethyl alcohol or water, and the mass ratio of the solvent to the ruthenium-based salicylic acid vinyl imidazole ionic liquid is 1-10: 1; the mixing is carried out by a homogenizer at the speed of 800-1000r/min for 4-5 min.
Further, in the step (2), the mass ratio of the divinylbenzene to the ruthenium-based salicylic acid vinyl imidazole ionic liquid is 1-10: 1.
further, in the step (2), the radical initiator is ammonium persulfate, and the mass ratio of the radical initiator to the ruthenium-based salicylic acid vinyl imidazole ionic liquid is 1: 200-500; the reaction time is 10-24h, and the temperature is 60-100 ℃; the drying temperature is 60-75 ℃, and the drying time is 10-12 h; the particle size of the solid powder is 1-1.5 μm.
Further, in the step (3), the solvent is water, and the mass-to-volume ratio of the solid powder to the solvent is 1: 10-100 g/ml.
Further, in the step (3), the reducing agent is sodium borohydride, and the mass ratio of the reducing agent to the solid powder is 1: 3-10; the reaction time is 0.6-2.0h, and the temperature is 60-120 ℃; the drying temperature is 60-100 ℃, and the drying time is 1-5 h; the particle size of the ruthenium-based metal ion liquid polymer microsphere artificial enzyme is 1-1.5 mu m.
An artificial enzyme of ruthenium-based metal ion liquid polymer microspheres prepared by the preparation method.
The application of the ruthenium-based metal ion liquid polymer microsphere artificial enzyme is characterized in that the ruthenium-based metal ion liquid polymer microsphere artificial enzyme is used for detecting prochloraz by a colorimetric method.
The application of the ruthenium-based metal ion liquid polymer microsphere artificial enzyme comprises the following specific application methods: the ruthenium-based metal ion liquid polymer microsphere is artificially enzymatically dispersed in water to prepare a dispersion liquid, the dispersion liquid is mixed with prochloraz standard solutions or sample solutions with different concentrations, the mixture is incubated for a period of time, then TMB solution and acetic acid buffer solution with the pH value of 4.0 are added to adjust the pH value, the mixture is shaken up and kept stand for 0.5 to 5min, and then the absorbance at the wavelength of 652nm is measured on a spectrophotometer.
Further, the pH value is adjusted to be 3.0-5.0, the incubation time is 15-35min, the incubation temperature is 35-60 ℃, and the concentration of the dispersion liquid is 1-5 mg/ml.
The invention obtains solid polymer microspheres through ionic liquid polymerization reaction, and the polymer monomer is ruthenium-based metal ionic liquid.
The beneficial technical effects of the invention are as follows:
(1) the ruthenium-based metal ionic liquid polymer microsphere artificial enzyme prepared by the method has a certain adsorption effect on TMB and oxygen, and the amphipathy of the ionic liquid polymer is favorable for combining TMB molecules with oxygen, so that the full contact between the oxygen and the TMB is promoted, and the electron transfer efficiency is improved; on the other hand, the ruthenium atom on the surface of the polymer microsphere has good dispersibility, and the agglomeration of ruthenium atoms in the catalytic process is prevented, so that higher catalytic activity is shown, the oxidation reaction of TMB is accelerated, and the absorbance at 652nm is obviously increased.
(2) The invention adopts metal ion liquid to prepare polymer microspheres through emulsion polymerization, and then ruthenium atoms on the microspheres are changed into a ruthenium simple substance through reduction, so that the artificial enzyme has oxidase activity and is obtained. The polymer artificial enzyme shows good dispersibility and high oxidase-like activity, and has a remarkable catalytic effect on the oxidation of 3,3 ', 5, 5' -tetramethylbenzidine to generate a blue product.
(3) The invention establishes a rapid, high-sensitivity and good-reproducibility colorimetric detection method for the prochloraz residue in the strawberry based on the inhibition effect of the prochloraz on the catalytic activity of the polymer microsphere artificial enzyme; the method has the detection limit of 8nM and the standard deviation of 3.25%, and has the advantages of low detection cost, simple operation, intuitive reading, no need of complicated sample pretreatment, no need of operation in a fixed laboratory by a skilled operator by using an expensive instrument, short detection time (only 30 min), and obviously lower detection limit than that of the existing method.
Drawings
FIG. 1 is an SEM image of an artificial enzyme of ruthenium-based metal ion liquid polymer microspheres prepared in example 1 of the present invention.
FIG. 2 is an XPS chart of an artificial enzyme of a ruthenium-based metal ionic liquid polymer microsphere prepared in example 1 of the present invention.
FIG. 3 is an absorption spectrum chart of the composite prepared in example 1 and comparative example 1 according to the present invention and the ruthenium nanoparticle of comparative example 2.
In the figure: a. ruthenium-based metal ionic liquid polymer microspheres prepared in example 1; b. the Co-His-GQD-G complex of comparative example 3; c. ruthenium nanoparticles prepared in comparative example 2; d. the metal-free ionic liquid polymer prepared in comparative example 1.
FIG. 4 is a graph showing the absorbance at 652nm of TMB solutions saturated with nitrogen, air and oxygen catalyzed by ruthenium-based metal ionic liquid polymer microspheres prepared in example 1 of the present invention.
FIG. 5 is a graph showing the relationship between the pH (A) of the reaction system, the incubation time (B), the incubation temperature (C) and the concentration of the artificial enzyme dispersion and the absorbance of the reaction system when applied to an example of the present invention.
FIG. 6 is an absorption spectrum of prochloraz at various concentrations measured by the artificial enzyme prepared in example 1 of the present invention.
FIG. 7 is a graph showing the relationship between the absorbance at 652nm and the concentration of prochloraz of the artificial enzyme prepared in example 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
Example 1
A ruthenium-based metal ion liquid polymer microsphere artificial enzyme is prepared by the following steps:
(1) 60mL of salicylic acid (0.1mol/L), 60mL of vinylimidazole (0.1mol/L) and 10mL of an aqueous solution of ruthenium trichloride (0.1mol/L) were mixed and stirred at 65 ℃ for 60min at 60r/min to obtain a black mixed solution. Then extracting with dichloromethane, carrying out rotary evaporation on the oil phase solution to remove the solvent, completely removing the solvent at the rotary evaporation temperature of 70 ℃ for 1h, and carrying out vacuum drying on the obtained solution at the temperature of 75 ℃ overnight to obtain black oily liquid, namely ruthenium-based salicylic acid vinyl imidazole ionic liquid.
(2) Dispersing 10g of ionic liquid in 100mL of water, adding 100g of divinylbenzene, mixing and homogenizing by using a homogenizer at a homogenizing speed of 1000r/min for 5min, adding 0.05g of ammonium persulfate free radical initiator after mixing uniformly, carrying out reflux reaction in a nitrogen atmosphere for 24h at a reaction temperature of 100 ℃, filtering and washing for 5 times by using deionized water, and drying at 75 ℃ for 12h to obtain powdery solid with the particle size of 1-1.5 microns;
(3) dispersing 10g of the powdery solid obtained in the step (2) in 100ml of water, adding 2g of sodium borohydride, reacting for 2h at 80 ℃, filtering, and drying for 1h at 100 ℃ to obtain the ruthenium-based metal ion liquid polymer microspheres, wherein the particle size of the artificial enzyme of the ruthenium-based metal ion liquid polymer microspheres is 1-1.5 mu m.
The artificial enzyme prepared in the embodiment is used for detecting a prochloraz sample, and the detection steps are as follows:
the ruthenium-based metal ion liquid polymer microsphere artificial enzyme prepared in the embodiment 1 of the invention is taken, 5mg/mL ruthenium-based metal ion liquid polymer microsphere dispersion (100 mu L) is prepared to be mixed with prochloraz standard solutions or sample solutions (200 mu L) with different concentrations, incubation is carried out at 50 ℃ for 30min, then 300 mu L TMB solution (10mmol/L) and 400 mu L acetic acid buffer solution (pH 4.0) are added, shaking is carried out, standing is carried out for 2min, and then scanning spectrum is carried out on a spectrophotometer or absorbance at the wavelength of 652nm is measured.
In the invention, the influence of different pH values, different incubation temperatures, different incubation times and artificial enzyme concentrations on absorbance (as shown in figure 5) in reaction is compared, and finally determined reaction conditions are as follows: pH4.0, incubation time of 30min, incubation temperature of 40 ℃, and artificial enzyme concentration of the polymer microspheres is 3 mg/mL.
Example 2:
a ruthenium-based metal ion liquid polymer microsphere artificial enzyme, the preparation method comprises the following steps:
(1) 60mL of salicylic acid (1.2mol/L), 50mL of vinylimidazole (1.2mol/L) and 15mL of an aqueous solution of ruthenium trichloride (1.2mol/L) were mixed and stirred at 75r/min at 65 ℃ for 45min to obtain a black mixed solution. Then extracting with dichloromethane, carrying out rotary evaporation on the oil phase solution to remove the solvent, completely removing the solvent at the rotary evaporation temperature of 70 ℃ for 1h, and carrying out vacuum drying on the obtained solution at the temperature of 70 ℃ for 8h to obtain black oily liquid, namely ruthenium-based salicylic acid vinyl imidazole ionic liquid.
(2) Dispersing 100g of ionic liquid in 100mL of water, adding 100g of divinylbenzene, mixing and homogenizing by using a homogenizer at the homogenizing speed of 800r/min for 5min, adding 0.2g of ammonium persulfate free radical initiator after mixing uniformly, carrying out reflux reaction in a nitrogen atmosphere for 10 hours at the reaction temperature of 60 ℃, filtering, washing for 3 times by using deionized water, and drying at the temperature of 60 ℃ for 10 hours to obtain powdery solid with the particle size of 1-1.5 micrometers;
(3) dispersing 1g of the powdery solid obtained in the step (2) in 100ml of water, adding 0.33g of sodium borohydride, reacting at 120 ℃ for 0.6h, filtering, and drying at 60 ℃ for 5h to obtain the ruthenium-based metal ion liquid polymer microspheres, wherein the particle size of the artificial enzyme of the ruthenium-based metal ion liquid polymer microspheres is 1-1.5 mu m.
Example 3:
a ruthenium-based metal ion liquid polymer microsphere artificial enzyme is prepared by the following steps:
(1) 60mL of salicylic acid (0.05mol/L), 60mL of vinylimidazole (0.05mol/L) and 10mL of an aqueous solution of ruthenium trichloride (0.05mol/L) were mixed and stirred at 60r/min at 65 ℃ for 55min to obtain a black mixed solution. Then extracting with dichloromethane, performing rotary evaporation on the oil phase solution to remove the solvent, completely removing the solvent at the rotary evaporation temperature of 70 ℃ for 1h, and performing vacuum drying on the obtained solution at 100 ℃ for 12h to obtain black oily liquid, namely ruthenium-based salicylic acid vinyl imidazole ionic liquid.
(2) Dispersing 20g of ionic liquid in 100mL of water, adding 100g of divinylbenzene, mixing and homogenizing by using a homogenizer at a homogenizing speed of 900r/min for 5min, adding 0.066g of ammonium persulfate free radical initiator after mixing uniformly, carrying out reflux reaction in a nitrogen atmosphere for 24h at a reaction temperature of 80 ℃, filtering and washing for 4 times by using deionized water, and drying at 70 ℃ for 11h to obtain powdery solid with the particle size of 1-1.5 microns;
(3) dispersing 10g of the powdery solid obtained in the step (2) in 100ml of water, adding 0.1g of sodium borohydride, reacting at 60 ℃ for 1h, filtering, and drying at 70 ℃ for 3h to obtain the ruthenium-based metal ion liquid polymer microspheres, wherein the particle size of the artificial enzyme of the ruthenium-based metal ion liquid polymer microspheres is 1-1.5 mu m.
Comparative example 1:
a metal-free ionic liquid polymer was prepared in the same manner as in example 1, except that no ruthenium trichloride solution was added.
Comparative example 2:
the preparation method of ruthenium nanoparticles is the same as that in example 1, but the method further comprises the following step of carrying out oxidative decomposition on the ruthenium-based metal ion liquid polymer microsphere artificial enzyme prepared in example 1 in the air to obtain ruthenium nanoparticles serving as comparative example 2.
Comparative example 3:
according to the article of Li N et al (Colorimetric detection of chlorination in sea based on benzene felt-graphene nanohybrid with excellent oxidase-like activity and reusability [ J]Journal of Hazardous Materials,2021,415:125752.) A Co-His-GQD-G complex was prepared as comparative example 3 by: dispersing 0.024g of graphene oxide in 50ml of deionized water, and adding 50ml of His-GOD solution of 6mg/ml and 0.3g of CTAC while stirring to formA His-GOD-GO composition; 1M NaOH was added to adjust the pH to 7.0, and 12ml of 0.1M CoCl was added 2 And (3) obtaining a Co-His-GQD-GO composition, centrifuging at 3000rpm for 5 minutes, washing for 3 times, drying at 80 ℃ for 4 hours, collecting precipitates, and reducing the precipitates at 600 ℃ in a nitrogen atmosphere for 2 hours to obtain a Co-His-GQD-G compound.
Test example:
(1) the artificial enzyme catalysis capability test of the ruthenium-based metal ion liquid polymer microsphere comprises the following steps:
in order to understand the mechanism of catalytic action, the catalytic behavior of the ruthenium-based metal ion liquid polymer microspheres in saturated solutions of nitrogen, air and oxygen is respectively examined. As can be seen from FIG. 4, the absorbance of the nitrogen saturated solution is close to zero, which indicates that the ruthenium-based metal ionic liquid polymer microspheres can not catalyze TMB to generate blue compounds in the absence of oxygen. The absorbance of the air saturated solution is obviously higher than that of the nitrogen saturated solution, which indicates that oxygen plays an important role in the reaction. Compared with the air saturated solution, the absorbance of the oxygen saturated solution is higher, which proves that the ruthenium-based metal ionic liquid polymer microspheres have better catalytic activity under the oxygen saturation condition, and the oxygen content of pure oxygen is higher than that of air. The facts also prove that the ruthenium-based metal ion liquid polymer microspheres have oxidase-like activity in TMB oxidation reaction, and the oxidase-like activity is probably derived from the catalytic capability of the ruthenium-based metal ion liquid polymer microspheres on oxygen activation to generate active oxygen.
(2) The method comprises the following steps of (1) manually detecting prochloraz by using ruthenium-based metal ion liquid polymer microspheres:
in order to research the influence of prochloraz on the activity of ruthenium-based metal ion liquid polymer microsphere artificial enzyme, 0.1mL of prochloraz is added into 0.1mL of 5mg/mL aqueous dispersion solution of ruthenium-based metal ion liquid polymer microsphere, the mixture is incubated at 50 ℃ for 30min, then 300 mu L of TMB solution (10mmol/L) and 400 mu L of acetic acid buffer solution (pH 4.0) are added, the mixture is shaken evenly and placed for 2min, then the catalytic capability of oxidation reaction of the TMB is inspected, and the absorbance at 652nm is measured. The result shows that under the same condition, the absorbance is obviously reduced compared with that without adding the prochloraz in the presence of the prochloraz, and the prochloraz is proved to be capable of effectively inhibiting the activity of the similar oxidase of the ruthenium-based metal ion liquid polymer microspheres.
(3) Example to comparative example catalytic performance comparison:
the catalytic performance of the materials prepared in example 1 and comparative examples 1-3, respectively, was examined using the blue compound formed by oxidation of TMB as a model reaction. The results are shown in FIG. 3. As can be seen from FIG. 3, in the presence of the metal-free ionic liquid polymer prepared in comparative example 1, the color development system is a colorless solution, the absorption spectrum has no absorption peak, is similar to a straight line, and has a small absorbance, which indicates that the metal-free ionic liquid polymer has no catalytic effect on the oxidation reaction of TMB. In the presence of the ruthenium nanoparticles prepared in the comparative example 2, the color development system is light blue, and a stronger absorption peak appears at 652nm, which indicates that the ruthenium nanoparticles have remarkable catalytic action. After ruthenium nanoparticles are replaced by the ruthenium-based metal ionic liquid polymer microspheres prepared in example 1, the absorbance of the system is rapidly increased, and the catalytic activity of the polymer microspheres is proved to be superior to that of the single ruthenium nanoparticles. As the ruthenium element on the ruthenium-based metal ionic liquid polymer microsphere is reduced into the ruthenium simple substance, the combination of the ruthenium nano particle and the ionic liquid polymer generates an obvious synergistic catalytic effect. In addition, the activity of the like oxidase of the ruthenium-based metal ionic liquid polymer microspheres is higher than that of the Co-His-GQD-G compound in the comparative example 3, because, on one hand, the polymer microspheres have certain adsorption effect on TMB and oxygen, and the amphipathy of the ionic liquid polymer is favorable for combining TMB molecules with oxygen, so that the full contact of the oxygen and the TMB is promoted, and the electron transfer efficiency is improved; on the other hand, the ruthenium atom on the surface of the polymer microsphere has good dispersibility, and the agglomeration of ruthenium atoms in the catalytic process is prevented, so that higher catalytic activity is shown, the oxidation reaction of TMB is accelerated, and the absorbance at 652nm is obviously increased.
(4) Establishing a standard equation of the prochloraz for artificial enzyme detection:
in order to verify the feasibility of the prochloraz detection method, the invention respectively measures the absorption spectrum and the absorbance at 652nm of a reaction system consisting of artificial enzyme and TMB (figures 6-7) under different prochloraz concentrations, and as shown in figure 6, as can be seen from figure 6, the absorbance of the reaction system is reduced along with the increase of the prochloraz concentration. This is because the higher the concentration of prochloraz, the stronger the inhibition to the catalytic activity of the ruthenium-based metal ion liquid polymer microsphere, resulting in slower oxidation reaction and lower absorbance of the reaction system. Further, fig. 7 shows that the absorbance linearly decreases with increasing concentration of prochloraz in the range of 10nM to 100 μ M. The correlation linear regression equation is a-0.08555 × C +0.7276 with a correlation coefficient of 0.977, where C is the concentration of prochloraz.
(5) Detection limit and repeatability test:
based on the 3-fold signal-to-dryness ratio, the detection limit of the artificial enzyme prepared in the embodiment of the invention for spectrophotometric detection of prochloraz is 8 nM. 30 prochloraz samples with the same concentration (10ng/ml) are taken as an example to verify the reproducibility of the method, the relative standard deviation of the experimental result is 3.25%, and the reproducibility is good; the linear range of detection of the invention is 0.01-100. mu.M. Compared with comparative example 3, the linear range is wider, and the detection line is increased from 5 μ M to 8 nM. Compared with the conventional liquid chromatography and gas chromatography, the method has a better linear range and a lower detection limit. The method is low in cost, simple to operate, intuitive in reading, free of complicated sample pretreatment, short in detection time which is only 30min and obviously lower in detection limit than that of the existing method, does not need to use biological enzyme, and the used ruthenium-based metal ion liquid polymer microspheres are simple to prepare and easy to store, so that the ruthenium-based metal ion liquid polymer microspheres have obvious advantages in detection of prochloraz in strawberries, and represent the potential of application in field rapid detection in quality safety supervision of base-level agricultural products.
FIG. 1 is an SEM image of an artificial enzyme of ruthenium-based metal ion liquid polymer microspheres prepared in example 1 of the present invention. FIG. 2 is an XPS chart of an artificial enzyme of a ruthenium-based metal ionic liquid polymer microsphere prepared in example 1 of the present invention. As can be seen from FIG. 1-2, the ruthenium-based metal ionic liquid polymer microspheres have uniform size, particle size of about 1 μm, and are spherical, and the ruthenium-based metal ionic liquid polymer microspheres are composed of carbon (C), ruthenium (Ru), oxygen (O) and nitrogen (N). FIG. 1C high resolution peak C1s XPS can be decomposed into C-C, C-O and C-N bondsIndicating that the oxygen and nitrogen groups in the ionic liquid polymer are not completely reduced. In addition, Ru 3d in XPS spectra 3/2 The peak overlaps with the peak of C1s, which affects the analysis of the chemical environment of the element, so the substitution of Ru 3p XPS peak, 462.5 is the characteristic of zero-valent ruthenium metal, which indicates that the ruthenium is reduced to the elemental state.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still make modifications to the technical solutions described in the foregoing embodiments, or make equivalent substitutions and improvements to part of the technical features of the foregoing embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of an artificial enzyme of ruthenium-based metal ion liquid polymer microspheres is characterized by comprising the following steps:
(1) mixing a salicylic acid solution, a vinyl imidazole solution and a ruthenium trichloride solution, stirring, extracting, evaporating the obtained oil phase solution to remove a solvent, and drying to obtain ruthenium-based salicylic acid vinyl imidazole ionic liquid;
(2) dispersing the ruthenium-based salicylic acid vinyl imidazole ionic liquid obtained in the step (1) in a solvent, adding divinylbenzene for mixing, adding a free radical initiator for reaction, separating precipitate, washing with water, and drying to obtain solid powder;
(3) dispersing the solid powder obtained in the step (2) in a solvent to obtain a reactant, adding a reducing agent for reaction, and drying the solid obtained by filtering to obtain the ruthenium-based metal ion liquid polymer microsphere artificial enzyme;
in the step (2), the free radical initiator is ammonium persulfate;
in the step (3), the reducing agent is sodium borohydride.
2. The preparation method according to claim 1, wherein in the step (1), the molar concentrations of the salicylic acid solution, the vinylimidazole solution and the ruthenium trichloride solution are all 0.05-1.2 mol/L; the volume ratio of the salicylic acid solution to the vinyl imidazole solution to the ruthenium trichloride solution is 6: 5-6: 0.5-1.5.
3. The preparation method according to claim 1, wherein in the step (1), the stirring speed is 60-80r/min, and the time is 45-60 min; the extracting agent used for extraction is dichloromethane; the evaporation is rotary evaporation; the drying temperature is 70-100 ℃, and the drying time is 8-14 h.
4. The preparation method according to claim 1, wherein in the step (2), the solvent is absolute ethyl alcohol or water, and the mass ratio of the solvent to the ruthenium-based salicylic acid vinyl imidazole ionic liquid is 1-10: 1; the mixing is carried out by a homogenizer at the speed of 800-1000r/min for 4-5 min.
5. The preparation method according to claim 1, wherein in the step (2), the mass ratio of the divinylbenzene to the ruthenium-based salicylic acid vinylimidazole ionic liquid is 1 to 10: 1.
6. the preparation method according to claim 1, wherein in the step (2), the mass ratio of the radical initiator to the ruthenium-based salicylic acid vinyl imidazole ionic liquid is 1: 200-500; the reaction time is 10-24h, and the temperature is 60-100 ℃; the drying temperature is 60-75 ℃, and the drying time is 10-12 h; the particle size of the solid powder is 1-1.5 mu m.
7. The preparation method according to claim 1, wherein in the step (3), the solvent is water, and the mass-to-volume ratio of the solid powder to the solvent is 1: 10-100 g/ml.
8. The production method according to claim 1, wherein in the step (3), the mass ratio of the reducing agent to the solid powder is 1: 3-10; the reaction time is 0.6-2.0h, and the temperature is 60-120 ℃; the drying temperature is 60-100 ℃, and the drying time is 1-5 h; the particle size of the ruthenium-based metal ion liquid polymer microsphere artificial enzyme is 1-1.5 mu m.
9. An artificial enzyme of ruthenium-based metal ion liquid polymer microspheres prepared by the preparation method of any one of claims 1 to 8.
10. The use of the artificial enzyme of ruthenium-based metal ion liquid polymer microspheres according to claim 9, wherein the artificial enzyme of ruthenium-based metal ion liquid polymer microspheres is used for colorimetric detection of prochloraz.
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