CN114570362A - Preparation method and application of nano enzyme - Google Patents
Preparation method and application of nano enzyme Download PDFInfo
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- CN114570362A CN114570362A CN202210205560.5A CN202210205560A CN114570362A CN 114570362 A CN114570362 A CN 114570362A CN 202210205560 A CN202210205560 A CN 202210205560A CN 114570362 A CN114570362 A CN 114570362A
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 38
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 136
- 239000010703 silicon Substances 0.000 claims abstract description 136
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims abstract description 112
- 238000003756 stirring Methods 0.000 claims abstract description 69
- -1 silicon amide Chemical class 0.000 claims abstract description 65
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims abstract description 48
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
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- JLHYHIKLLHOPOB-UHFFFAOYSA-N [Si][Au][Pt] Chemical compound [Si][Au][Pt] JLHYHIKLLHOPOB-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 229910000033 sodium borohydride Inorganic materials 0.000 claims abstract description 14
- 239000012279 sodium borohydride Substances 0.000 claims abstract description 14
- 239000001509 sodium citrate Substances 0.000 claims abstract description 14
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 14
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- 239000002244 precipitate Substances 0.000 claims description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
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- 238000000034 method Methods 0.000 claims description 21
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 20
- 239000007864 aqueous solution Substances 0.000 claims description 18
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 17
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 claims description 17
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 17
- 229960004025 sodium salicylate Drugs 0.000 claims description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- 238000005119 centrifugation Methods 0.000 claims description 15
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 claims description 14
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- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 abstract description 12
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- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
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- WGYKZJWCGVVSQN-UHFFFAOYSA-N propylamine Chemical group CCCN WGYKZJWCGVVSQN-UHFFFAOYSA-N 0.000 description 1
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- YUYCVXFAYWRXLS-UHFFFAOYSA-N trimethoxysilane Chemical compound CO[SiH](OC)OC YUYCVXFAYWRXLS-UHFFFAOYSA-N 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
- B01J23/38—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of noble metals
- B01J23/48—Silver or gold
- B01J23/52—Gold
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J35/00—Catalysts, in general, characterised by their form or physical properties
- B01J35/40—Catalysts, in general, characterised by their form or physical properties characterised by dimensions, e.g. grain size
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J37/00—Processes, in general, for preparing catalysts; Processes, in general, for activation of catalysts
- B01J37/0009—Use of binding agents; Moulding; Pressing; Powdering; Granulating; Addition of materials ameliorating the mechanical properties of the product catalyst
- B01J37/0018—Addition of a binding agent or of material, later completely removed among others as result of heat treatment, leaching or washing,(e.g. forming of pores; protective layer, desintegrating by heat)
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J37/00—Processes, in general, for preparing catalysts; Processes, in general, for activation of catalysts
- B01J37/16—Reducing
- B01J37/18—Reducing with gases containing free hydrogen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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Abstract
The invention discloses a preparation method and application of nano enzyme, wherein the preparation method of the nano enzyme comprises the following steps: taking a cationic surfactant as a template, adding a silicon source, a catalyst, a structure directing agent and an organic solvent, and reacting to obtain silicon spheres; carrying out amination treatment on the silicon spheres to obtain silicon amide spheres; dispersing the silicon amide spheres in water, adding the gold dispersion liquid, and stirring to obtain a silicon sphere-nanogold compound; and dispersing the silicon sphere-nano gold compound in water, adding a platinum-containing solution and a sodium citrate solution, stirring, adding a sodium borohydride solution, and stirring for reaction to obtain the gold-platinum-silicon-based nano enzyme. The nano enzyme prepared by the invention has specific recognition on mercury ions, can be used for detecting the content of the mercury ions, and has a certain positive effect on the optimization and upgrading of a mercury ion detection system in the future.
Description
Technical Field
The invention relates to the field of nano materials, in particular to a preparation method and application of nano enzyme.
Background
Mercury ions have high toxicity, can cause serious damage to human bodies through food and water pollution, and even at ultra-low concentrations, mercury and its compounds have various hazards to human health. The mercury ions accumulated in the human body are difficult to be discharged through the metabolism of the human body. Trace amounts of mercury ions can cause toxicity to the liver, kidneys and other organs, leading to oral inflammation, tremors of the hands, neurological disorders and chronic mercury poisoning. Therefore, rapid and sensitive detection of mercury ions is important.
Traditional mercury ion detection methods include atomic absorption spectrometry, inductively coupled plasma mass spectrometry, atomic fluorescence spectrometry, and the like, and most of these methods are limited to laboratories with complete equipment due to heavy and expensive equipment and cumbersome sample preparation steps, so that they cannot be used for on-site analysis applications.
Disclosure of Invention
The invention mainly aims to provide a preparation method and application of nano enzyme, and aims to provide the nano enzyme with strong anti-interference performance, wherein the nano enzyme can be applied to the rapid detection of mercury ions.
In order to achieve the purpose, the invention provides a preparation method of nano enzyme and application thereof, wherein the preparation method of the nano enzyme comprises the following steps:
taking a cationic surfactant as a template, adding a silicon source, a catalyst, a structure directing agent and an organic solvent, and reacting to obtain silicon spheres;
carrying out amination treatment on the silicon spheres to obtain silicon amide spheres;
dispersing the silicon amide spheres in water, adding the gold dispersion liquid, and stirring to obtain a silicon sphere-nanogold compound;
and dispersing the silicon sphere-nano gold compound in water, adding a platinum-containing solution and a sodium citrate solution, stirring, adding a sodium borohydride solution, and stirring for reaction to obtain the gold-platinum-silicon-based nano enzyme. Optionally, taking a cationic surfactant as a template, adding a silicon source, a catalyst and a structure directing agent, and reacting to obtain silicon spheres:
the cationic surfactant comprises cetyl trimethyl ammonium bromide; and/or the presence of a gas in the gas,
the silicon source comprises tetraethoxysilane; and/or the presence of a gas in the gas,
the catalyst comprises triethanolamine; and/or the presence of a gas in the gas,
the structure directing agent comprises sodium salicylate.
Optionally, taking a cationic surfactant as a template, adding a silicon source, a catalyst and a structure directing agent, and reacting to obtain the silicon spheres, wherein the step of reacting to obtain the silicon spheres comprises the following steps:
adding triethanolamine into water, and stirring at 80 deg.C for 30-60min to obtain solution A;
adding cetyl trimethyl ammonium bromide and sodium salicylate into the solution A, and stirring at constant temperature for 1h to obtain a solution B;
adding tetraethoxysilane into the solution B, stirring at constant temperature, and reacting for 12-15h to obtain a reaction solution C;
centrifuging and washing the reaction solution C to remove redundant reactants to obtain a precipitate, dispersing the precipitate in a mixed solution of hydrochloric acid and methanol, and stirring at 60 ℃ for 6-10h to obtain a solution D;
and centrifuging and washing the solution D to remove redundant reactants to obtain a precipitate, re-dispersing the obtained precipitate in ethanol, and placing the ethanol at a temperature lower than room temperature for later use to obtain the silicon spheres.
Optionally, the step of performing amination treatment on the silicon spheres to obtain aminated silicon spheres includes:
adding (3-aminopropyl) trimethoxy silane into the silicon spheres, and stirring, centrifuging and washing to obtain the silicon amide spheres.
Optionally, in the step of performing amination treatment on the silicon spheres to obtain aminated silicon spheres, the volume ratio of the silicon source to (3-aminopropyl) trimethoxysilane is 1: (24-28).
Optionally, in the step of performing amination treatment on the silicon spheres to obtain aminated silicon spheres, the stirring time is 5-7 hours; and/or the presence of a gas in the gas,
the rotation speed of the centrifugation is 5000-; and/or the presence of a gas in the gas,
the centrifugation time is 4-10 min.
Optionally, in the step of dispersing the silicon amide spheres in water, adding the gold dispersion liquid, and stirring to obtain the silicon sphere-nanogold composite, adding (120-.
Optionally, dispersing the silicon sphere-nanogold composite in water, adding a platinum-containing solution and a sodium citrate solution, stirring, adding a sodium borohydride solution, and stirring to react to obtain the gold-platinum-silicon-based nanoenzyme, wherein the platinum-containing solution comprises H2PtCl6An aqueous solution.
Optionally, in the step of dispersing the silicon sphere-nanogold composite in water, adding a platinum-containing solution and a sodium citrate solution, stirring, adding a sodium borohydride solution, and stirring to react to obtain the gold-platinum-silicon-based nanoenzyme, adding (8-9) mmoL platinum to the silicon sphere-nanogold composite prepared by reacting every 2mL of silicon source.
Optionally, the invention further provides an application of the nano enzyme prepared by the preparation method, and the nano enzyme is used for detecting mercury ions.
According to the technical scheme, the nano enzyme takes silicon as a raw material to prepare nano silicon spheres, and on the basis, the nano enzyme with enzyme-like properties is prepared by carrying nano gold and nano platinum on static electricity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a schematic flow chart of an embodiment of a method for preparing nanoenzymes according to the present invention;
FIG. 2 is a scanning electron micrograph of dendritic silicon spheres prepared in example 1 of the present invention;
FIG. 3 is a transmission electron microscope image of the silicon sphere-nanogold composite prepared in example 1 of the invention;
FIG. 4 is a transmission electron micrograph of nanoenzyme prepared in example 1 of the present invention;
FIG. 5 is a TEM image of dendritic silica spheres, an element mapping image of silica sphere-nanogold composite particles, an element mapping image of gold-platinum-silicon-based nanoenzyme particles and an element mapping image of silica spheres prepared in example 1 of the present invention;
FIG. 6 is the UV spectra of silicon spheres, silicon amide spheres, gold ions, silicon sphere-nanogold complexes and gold-platinum-silicon-based nanoenzymes prepared in example 1 of the present invention;
FIG. 7 is Zeta potential diagram of silicon spheres, silicon amide spheres, gold ions, silicon sphere-gold nanoparticles complexes and gold-platinum-silicon-based nanoenzymes prepared in example 1 of the present invention;
FIG. 8 is a TEM image and elemental mapping images of gold, platinum, silicon, mercury, and oxygen elements of gold-platinum-silicon-based nanoenzyme prepared in example 1 of the present invention;
FIG. 9 is the ultraviolet absorbance and color development of the reaction of the gold-platinum-silicon-based nanoenzyme prepared in example 1 of the present invention with mercury ions and a series of interfering ions.
FIG. 10 is a linear relationship diagram of UV absorbance and concentration of the negative Au-Pt-Si-based nanoenzyme prepared in example 1 of the present invention applied to mercury ion detection.
The implementation, functional features and advantages of the present invention will be further described with reference to the accompanying drawings.
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, if directional indications (such as upper, lower, left, right, front, rear, outer and inner … …) are involved in the embodiment of the present invention, the directional indications are only used to explain the relative position relationship between the components, the motion situation, etc. in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indications are changed accordingly.
In addition, if there is a description of "first", "second", etc. in an embodiment of the present invention, the description of "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, the meaning of "and/or" appearing throughout includes three juxtapositions, exemplified by "A and/or B", including either A or B or both A and B. In addition, technical solutions between the embodiments may be combined with each other, but must be based on the realization of the technical solutions by a person skilled in the art, and when the technical solutions are contradictory to each other or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
Mercury ions have high toxicity, can cause serious damage to human bodies through food and water pollution, and even at ultra-low concentrations, mercury and its compounds have various hazards to human health. The mercury ions accumulated in the human body are difficult to be discharged through the metabolism of the human body. Trace mercury ions can cause toxicity to the liver, kidneys and other organs, leading to oral inflammation, tremors of the hands, neurological disorders and chronic mercury poisoning. Therefore, rapid and sensitive detection of mercury ions is important.
Traditional mercury ion detection methods include atomic absorption spectrometry, inductively coupled plasma mass spectrometry, atomic fluorescence spectrometry, and the like, and most of these methods are limited to laboratories with complete equipment due to heavy and expensive equipment and cumbersome sample preparation steps, so that they cannot be used for on-site analysis applications.
In the technical scheme of the invention, the nano enzyme is mainly prepared by taking nano silicon as a carrier and loading nano gold particles and nano platinum particles through electrostatic action, the nano enzyme has a specific recognition effect on mercury ions, can be used for qualitative detection of the mercury ions and can quickly recognize whether the mercury ions exist, a certain amount of mercury ions are added into the prepared nano enzyme, and the absorbance is detected through an ultraviolet spectrophotometry, so that a mercury ion standard curve can be constructed, the method is convenient and reliable for later detection of the content of the mercury ions, can be used for field analysis, and has wide application prospects in the fields of environmental detection, disease diagnosis and the like.
In view of this, the present invention provides a method for preparing nanoenzymes and applications thereof, and aims to provide a method for rapidly detecting mercury ions with high sensitivity, and in the accompanying drawings, fig. 1 is a schematic flow diagram of an embodiment of the method for preparing nanoenzymes according to the present invention.
Referring to fig. 1, the preparation method of the nanoenzyme includes the following steps:
and step S10, taking the cationic surfactant as a template, adding a silicon source, a catalyst, a structure directing agent and an organic solvent, and reacting to obtain the silicon spheres.
In this embodiment, the first step of preparing the nanoenzyme is to prepare a silicon sphere, which can be prepared by various methods, so as to ensure that the prepared silicon sphere has a cavity with a certain size for other ions to attach.
In one embodiment, the silicon spheres are three-dimensional dendritic silicon spheres with a substitution and central radial mesoporous channel obtained by a one-pot two-phase continuous growth method by taking a cationic surfactant cetyl trimethyl ammonium bromide (CTAC) as a template, Tetraethoxysilane (TEOS) as a silicon source, Triethanolamine (TEA) as a catalyst and sodium salicylate as a structure directing agent in combination with an organic solvent.
The method comprises the following steps of taking a cationic surfactant as a template, adding a silicon source, a catalyst and a structure directing agent, and reacting to obtain silicon spheres:
adding triethanolamine into water, and stirring at 80 deg.C for 30-60min to obtain solution A;
adding cetyl trimethyl ammonium bromide and sodium salicylate into the solution A, and stirring at constant temperature for 1h to obtain a solution B;
adding tetraethoxysilane into the solution B, stirring at constant temperature, and reacting for 12-15h to obtain a reaction solution C;
centrifuging and washing the reaction solution C to remove redundant reactants to obtain a precipitate, dispersing the precipitate in a mixed solution of hydrochloric acid and methanol, and stirring at 60 ℃ for 6-10h to obtain a solution D;
and centrifuging and washing the solution D to remove redundant reactants to obtain a precipitate, re-dispersing the obtained precipitate in ethanol, and placing the ethanol at a temperature lower than room temperature for later use to obtain the silicon spheres.
Adding triethanolamine into water, and stirring for 30-60min (such as 30min, 40min, 50min, and 60 min), preferably 30min to ensure uniform dispersion of triethanolamine in water; cetyl trimethyl ammonium bromide and sodium salicylate are stirred for 1 hour at the temperature of 80 ℃, the reagents are completely mixed, the cetyl trimethyl ammonium bromide is used as a mesoporous template to play a role in adjusting the aperture, the sodium salicylate is used as a structure guiding agent and has a hole expanding function, and the cetyl trimethyl ammonium bromide and the sodium salicylate jointly act to prepare the nano silicon with uniform and proper particle size; adding ethyl orthosilicate as a silicon source, stirring at constant temperature for 12-15h, such as 12h, 13h, 14h and 15h, preferably for 12h, and obtaining the nano silicon spheres with good stability and size meeting the requirement; stirring for 6-10h, such as 6h, 7h, 8h, 9h and 10h, preferably for 6h, at 60 ℃ by using a mixed solution of hydrochloric acid and methanol, wherein the stirring time is fully stirring for completely removing reactants inside and outside the pore diameter of the nano silicon spheres; the stirred solution is further centrifugally washed for a plurality of times to remove redundant acid, so that high-purity nano silicon balls can be obtained, and the subsequent treatment process is not influenced.
And step S20, performing amination treatment on the silicon spheres to obtain silicon amide spheres.
In order to ensure that the nano silicon spheres have stable adsorption effect and good wrapping property and avoid leakage of metal nano particles, the silicon spheres need to be aminated, in one embodiment of the invention, (3-aminopropyl) trimethoxysilane is added into the silicon spheres, and the silicon spheres are stirred at room temperature, centrifuged, washed and precipitated by ethanol and then dispersed in the ethanol to prepare the aminated silicon spheres.
The volume ratio of the silicon source to the (3-aminopropyl) trimethoxysilane is 1: (24-28), adding a (3-aminopropyl) trimethoxysilane pure sample into the silicon spheres to aminate the surfaces of the silicon spheres, wherein the excessive amount of the 3-aminopropyl) trimethoxysilane can cause waste of the (3-aminopropyl) trimethoxysilane, and the insufficient amination of the surfaces of the silicon spheres can cause insufficient amination, so that the silicon spheres can adsorb metal ions more efficiently in the later period, ensure a proper volume ratio and facilitate smooth reaction, preferably, the volume ratio of the silicon source to the (3-aminopropyl) trimethoxysilane is 1:25, the reaction is complete, and the added reagents are hardly wasted.
Adding (3-aminopropyl) trimethoxy silane into the silicon spheres, stirring at room temperature, centrifuging, washing the precipitate with ethanol, dispersing in ethanol, and preparing aminated silicon spheres, wherein the stirring time is 5-7h, for example, 5h, 6h, 7h, preferably 6 h; the rotation speed of the centrifugation is 5000-; the centrifugation time is 4-10min, for example, 4min, 5min, 6min, 7min, 8min, 9min, 10min, preferably 5min, in the process of preparing silicon amide spheres by using the silicon spheres, the silicon spheres are fully stirred to ensure full amination, and the aminated silicon spheres are completely separated by the centrifugation process; and (3) washing for multiple times to remove redundant reactants, wherein the prepared silicon amide spheres have high purity and contain no impurities.
And step S30, dispersing the silicon amide spheres in water, adding the gold dispersion liquid, and stirring to obtain the silicon sphere-nanogold composite.
According to the principle that an alkyl silane medium can be coated with silicon to prevent metal nanoparticles from falling off, silicon amide spheres are dispersed in water, gold dispersion liquid is added, stirring is carried out to obtain a silicon sphere-nanogold composite, wherein (120) -122 nmoL gold is added into every 2mL of silicon source silicon amide spheres obtained through reaction, preferably, 120.8nmoL gold is added into every 2mL of silicon source silicon amide spheres obtained through reaction, red silicon sphere-nanogold composite is obtained through stirring, the silicon amide spheres fully adsorb sufficient gold, the added gold is ensured not to be lost, the silicon spheres and the gold particles are subjected to electrostatic interaction, gold nanoparticles are adsorbed to the surfaces of the silicon spheres, and the size of the silicon spheres is basically not changed, so that the performance of the silicon sphere-nanogold after reaction is more stable.
And step S40, dispersing the silicon sphere-nanogold composite in water, adding a platinum-containing solution and a sodium citrate solution, stirring, adding a sodium borohydride solution, and stirring for reaction to obtain the gold-platinum-silicon-based nanoenzyme.
In order to make the nano silicon spheres specifically recognize mercury ions, further modification needs to be carried out on the silicon sphere-nano gold compound. In one embodiment of the present invention, H is added under the condition of vigorous stirring of the silicon sphere-nano gold composite2PtCl6The solution and the sodium citrate solution are stirred and added with NaBH which is kept in ice bath for standby4And continuously stirring the solution, standing the solution until the brown color of the solution is changed into black, centrifuging the solution, washing the solution with ultrapure water for three times, and finally dispersing the solution in the ultrapure water to obtain the nano enzyme. NaBH4The solution can well reduce H2PtCl6The solution is used for obtaining platinum, the reaction is easy to operate, the controllability is good, and NaBH which is quickly added after ice bath is adopted4Solution, guaranteed NaBH4The performance is not inactivated, the sodium citrate solution is used as a protective agent, the prepared nano metal particles are stable in size, the reaction conversion rate is high, the operability is strong, and the prepared nano enzyme is stable in property and not easy to be affected; the proportion of the platinum content to the gold-nano silicon compound is that (8-9) mmoL platinum is correspondingly added to the silicon ball-nano gold compound prepared by every 2mL of silicon source reaction, the gold-nano silicon compound can adsorb platinum as much as possible, and the obtained gold-platinum-silicon-based nanoenzyme is stable in performance, high in purity and multiple in adsorption sites.
Moreover, the invention also provides application of the nano enzyme prepared by the preparation method, and the nano enzyme is applied to detection of mercury ions.
Particularly, the nano enzyme has excellent physical properties, can specifically recognize mercury ions, and can be used for detecting the mercury ions.
Specifically, the method for detecting mercury ions by using the nano-enzyme comprises the following steps:
1. preparing nano enzyme;
2. preparing acetic acid buffer solution and hydrogen peroxide (H)2O2) A solution, 3',5,5' -Tetramethylbenzidine (TMB) solution;
3. mixing the prepared acetic acid buffer solution and hydrogen peroxide solution (H)2O2) Adding nano enzyme into an enzyme label plate, adding a mercury ion standard solution, finally adding a TMB solution, standing for 20min until the color is completely developed, putting the enzyme label plate into an ultraviolet spectrophotometer, and detecting the corresponding absorbance at the wavelength of 650 nm; taking the concentration of mercury ions as a horizontal coordinate and ultraviolet absorbance as a vertical coordinate, and performing linear fitting to obtain a standard curve and a standard curve equation;
4. mixing the prepared acetic acid buffer solution and hydrogen peroxide solution (H)2O2) Adding the nanoenzyme into an enzyme label plate, adding a sample to be detected, finally adding a TMB solution, standing for 20min until the color development is complete, putting the enzyme label plate into an ultraviolet spectrophotometer, and detecting the corresponding absorbance at the wavelength of 650 nm;
5. and (4) substituting the absorbance of the unknown solution into a corresponding standard curve equation, and converting the concentration of the mercury ions of the unknown solution.
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.
Example 1
(1) And (3) synthesizing dendritic silicon spheres:
first 0.068g Triethanolamine (TEA) was added to 25ml water and stirred at 80 ℃ for 30min, followed by 0.380g cetyltrimethylammonium bromide (CTAB) and 0.168g sodium salicylate (sodium salicylate), and stirring was continued for 1 hour at constant temperature; subsequently, 4mL of tetraethyl orthosilicate (TEOS) was added to the reaction mixture (TEOS was added rapidly over 1 min) and stirring was continued at 80 ℃ for 12 h. The resulting solution was washed at least three times with ethanol centrifugation to remove excess reagents. The precipitate obtained from the last centrifugation was dispersed in a mixed solution of 30mL of hydrochloric acid and 30mL of methanol, and stirred continuously at 60 ℃ for 6 hours, and then the reaction solution was centrifuged and washed, and the precipitate was redispersed in 100mL of ethanol and kept at a temperature lower than room temperature for further use.
(2) 400. mu.L of the silica spheres synthesized in step (1) was added with 400. mu.L of (3-aminopropyl) trimethoxysilane (APTES), and the mixture was stirred at room temperature for 6 hours. The stirred solution was centrifuged at 8000rpm for 5min, and the centrifuged lower precipitate was washed with ethanol and washed three times. Finally, the mixture was dispersed in 10mL of ethanol to prepare aminated silica spheres.
(3) And (3) centrifuging 600 mu L of aminated silicon balls prepared in the step (2), washing for 1 time by using ultrapure water, blow-drying redundant ethanol, ultrasonically dispersing in 2mL of ultrapure water after washing, and transferring to a penicillin bottle. Adding 5mL11.6nmoL/L of Au aqueous dispersion into a penicillin bottle, stirring at room temperature for 10min to obtain a red silicon ball nano-gold compound, centrifuging, washing with ultrapure water for 3 times for later use, ultrasonically dispersing in 2mL of ultrapure water, and transferring to a clean penicillin bottle for later use.
(4) Preparation of NaBH4Aqueous solution (0.1mol/L) and ice bath for standby. Violently stirring the silicon spheres and the nanogold compound in the step (3) at room temperature, and adding 0.2mL of H into the solution in the penicillin bottle2PtCl6Mixing the aqueous solution (19.3mmol/L) and 1mL of 1% (wt) sodium citrate aqueous solution, stirring for 15min, and finally rapidly adding 1mL of NaBH prepared for standby in ice bath4The aqueous solution was stirred for 15 min. Standing for 1h, centrifuging until the solution turns black from brown, washing with ultrapure water, repeating for 3 times, and finally dispersing in 2mL of ultrapure water to obtain the prepared nano enzyme.
Example 2
First 0.068g Triethanolamine (TEA) was added to 25ml water and stirred at 80 ℃ for 60min, followed by 0.380g cetyltrimethylammonium bromide (CTAB) and 0.168g sodium salicylate (sodium salicylate), and stirring was continued for 1 hour at constant temperature; subsequently, 4mL of tetraethyl orthosilicate (TEOS) was added to the reaction mixture (TEOS was added rapidly over 1 min) and stirring was continued at 80 ℃ for 15 h. The resulting solution was washed at least three times with ethanol centrifugation to remove excess reagents. The precipitate obtained from the last centrifugation was dispersed in a mixed solution of 30mL of hydrochloric acid and 30mL of methanol, and stirred continuously at 60 ℃ for 10 hours, and then the reaction solution was centrifuged and washed, and the precipitate was redispersed in 100mL of ethanol and left at a temperature lower than room temperature for further use.
(2) 400. mu.L of the silica spheres synthesized in step (1) was added with 448. mu.L of (3-aminopropyl) trimethoxysilane (APTES), and the mixture was stirred at room temperature for 6 hours. The stirred solution was centrifuged at 9000rpm for 4min, and the centrifuged lower precipitate was washed with ethanol and washed three times. Finally, the mixture was dispersed in 10mL of ethanol to prepare aminated silica spheres.
(3) And (3) centrifuging 600 mu L of aminated silicon balls prepared in the step (2), washing for 1 time by using ultrapure water, blow-drying redundant ethanol, ultrasonically dispersing in 2mL of ultrapure water after washing, and transferring to a penicillin bottle. Adding 5.05mL11.6nmoL/L of Au aqueous dispersion into a penicillin bottle, stirring for 10min at room temperature to obtain a red silicon ball nano-gold compound, centrifuging, washing for 3 times by using ultrapure water for later use, ultrasonically dispersing in 2mL of ultrapure water, and transferring to a clean penicillin bottle for later use.
(4) Preparation of NaBH4Aqueous solution (0.1mol/L) and ice bath for standby. Violently stirring the silicon spheres and the nanogold compound in the step (3) at room temperature, and adding 0.22mL of H into the solution in the penicillin bottle2PtCl6Mixing the aqueous solution (19.3mmol/L) with 1mL of 1% (wt) sodium citrate aqueous solution, stirring for 15min, and rapidly adding 1mL of NaBH prepared for use in ice bath4The aqueous solution was stirred for 15 min. Standing for 1h, centrifuging until the solution turns black from brown, washing with ultrapure water, repeating for 3 times, and finally dispersing in 2mL of ultrapure water to obtain the prepared nano enzyme.
Example 3
First 0.068g Triethanolamine (TEA) was added to 25ml water and stirred at 80 ℃ for 40min, followed by 0.380g cetyltrimethylammonium bromide (CTAB) and 0.168g sodium salicylate (sodium salicylate), and stirring was continued for 1 hour at constant temperature; subsequently, 4mL of Tetra Ethyl Ortho Silicate (TEOS) was added to the reaction mixture (TEOS was added rapidly over 1 min) and stirring was continued for 14h at 80 ℃. The resulting solution was washed at least three times with ethanol centrifugation to remove excess reagents. The precipitate obtained from the last centrifugation was dispersed in a mixed solution of 30mL of hydrochloric acid and 30mL of methanol, and stirred continuously at 60 ℃ for 9 hours, and then the reaction solution was centrifuged and washed, and the precipitate was redispersed in 100mL of ethanol and left at a temperature lower than room temperature for further use.
(2) 400. mu.L of the silica spheres synthesized in step (1) was taken, 390. mu.L of (3-aminopropyl) trimethoxysilane (APTES) was added thereto, and the mixture was stirred at room temperature for 7 hours. The stirred solution was centrifuged at 7000rpm for 8min, and the centrifuged lower precipitate was washed with ethanol and repeated three times. Finally, the mixture was dispersed in 10mL of ethanol to prepare aminated silica spheres.
(3) And (3) centrifuging 600 mu L of aminated silicon balls prepared in the step (2), washing for 1 time by using ultrapure water, blow-drying redundant ethanol, ultrasonically dispersing in 2mL of ultrapure water after washing, and transferring to a penicillin bottle. Adding 5.01mL11.6nmoL/L of Au aqueous dispersion into a penicillin bottle, stirring for 10min at room temperature to obtain a red silicon ball nano-gold compound, centrifuging, washing with ultrapure water for 3 times for later use, ultrasonically dispersing in 2mL of ultrapure water, and transferring to a clean penicillin bottle for later use.
(4) Preparation of NaBH4Aqueous solution (0.1mol/L) and ice bath for standby. Violently stirring the silicon spheres and the nanogold compound in the step (3) at room temperature, and adding 0.21mL of H into the solution in the penicillin bottle2PtCl6Mixing the aqueous solution (19.3mmol/L) with 1mL of 1% (wt) sodium citrate aqueous solution, stirring for 15min, and rapidly adding 1mL of NaBH prepared for use in ice bath4The aqueous solution was stirred for 15 min. Standing for 1h, centrifuging until the solution turns black from brown, washing with ultrapure water, repeating for 3 times, and finally dispersing in 2mL of ultrapure water to obtain the prepared nano enzyme.
Example 4
First 0.068g Triethanolamine (TEA) was added to 25ml water and stirred at 80 ℃ for 50min, followed by 0.380g cetyltrimethylammonium bromide (CTAB) and 0.168g sodium salicylate (sodium salicylate), and stirring was continued for 1 hour at constant temperature; subsequently, 4mL of tetraethyl orthosilicate (TEOS) was added to the reaction mixture (TEOS was added rapidly over 1 min) and stirring was continued at 80 ℃ for 13 h. The resulting solution was washed at least three times with ethanol centrifugation to remove excess reagents. The precipitate obtained from the last centrifugation was dispersed in a mixed solution of 30mL of hydrochloric acid and 30mL of methanol, and stirred continuously at 60 ℃ for 8 hours, and then the reaction solution was centrifuged and washed, and the precipitate was redispersed in 100mL of ethanol and left at a temperature lower than room temperature for further use.
(2) 400. mu.L of the silica spheres synthesized in step (1) was taken, and 420. mu.L of (3-aminopropyl) trimethoxysilane (APTES) was added thereto and stirred at room temperature for 6 hours. Centrifuging the stirred solution for 10min at 5000rpm, washing the centrifuged lower layer precipitate with ethanol, and repeating the washing three times. Finally, the mixture was dispersed in 10mL of ethanol to prepare aminated silica spheres.
(3) And (3) centrifuging 600 mu L of aminated silicon balls prepared in the step (2), washing with ultrapure water for 1 time, drying redundant ethanol, ultrasonically dispersing in 2mL of ultrapure water after washing, and transferring to a penicillin bottle. Adding 4.97mL11.6nmoL/L of Au aqueous dispersion into a penicillin bottle, stirring for 10min at room temperature to obtain a red silicon ball nano-gold compound, centrifuging, washing with ultrapure water for 3 times for later use, ultrasonically dispersing in 2mL of ultrapure water, and transferring to a clean penicillin bottle for later use.
(4) Preparation of NaBH4Aqueous solution (0.1mol/L) and ice bath for standby. Violently stirring the silicon spheres and the nanogold compound in the step (3) at room temperature, and adding 0.20mL of H into the solution in the penicillin bottle2PtCl6Mixing the aqueous solution (19.3mmol/L) with 1mL of 1% (wt) sodium citrate aqueous solution, stirring for 15min, and rapidly adding 1mL of NaBH prepared for use in ice bath4The aqueous solution was stirred for 15 min. Standing for 1h, centrifuging until the solution turns black from brown, washing with ultrapure water, repeating for 3 times, and dispersing in 2mL ultrapure water to obtain the final productThe nanoenzyme of (1).
Performance detection
1. Scanning electron microscope analysis is performed on the dendritic silicon spheres prepared in the step 1 of the example 1, and the result is shown in fig. 2, and as can be seen from fig. 2, the silicon spheres prepared in the step 1 of the example 1 have cavities with a certain size, and can be attached by other ions.
2. Scanning electron microscope analysis is performed on the gold-nano silicon sphere composite particles prepared in the step 3 of the example 1, the result is shown in fig. 3, and as can be seen from fig. 3, through the electrostatic interaction between the silicon amide spheres and the gold nanoparticles, the gold nanoparticles are adsorbed on the surface of the silicon spheres, and the size of the silicon spheres is not changed basically.
3. Scanning electron microscope analysis is carried out on the nanoenzyme prepared in the step 4 of the example 1, and the result is shown in figure 4, gold nanoparticles and platinum nanoparticles are loaded on the surface of the silicon spheres through electrostatic interaction to form a final gold-platinum-silicon-based nanoenzyme material, and the electron microscope figure shows that the nanoparticles are successfully loaded on the silicon spheres without changing the size of a sample basically.
4. TEM analysis and element mapping analysis are carried out on the silicon spheres obtained in the step 1 in the embodiment 1, and the results of the element mapping analysis on the gold-nano silicon sphere compound particles and the gold-platinum-silicon-based nanoenzyme particles obtained in the steps 3 and 4 in the embodiment 1 are shown in a figure 5.
5. Ultraviolet analysis was performed on the silicon spheres obtained in step 1, the silicon amide spheres obtained in step 2, the gold-nano silicon sphere composite obtained in step 3, the gold-platinum-silicon-based nanoenzyme loaded with gold and platinum nanoparticles obtained in step 4, and gold ions, and the results are shown in fig. 6, which indicates that the gold-loaded silicon spheres exhibited cash-like characteristic peaks, which disappeared after platinum loading, gold was covered with platinum, and the synthesis of the gold-platinum-silicon-based nanoenzyme was successful.
6. Zeta potential detection is carried out on the silicon spheres obtained in the step 1, the silicon amide spheres obtained in the step 2, the silicon sphere-nanogold compound obtained in the step 3, the gold-platinum-silicon-based nanoenzyme obtained in the step 4 and gold ions in the example 1, and the results are shown in a figure 7.
7. TEM detection is carried out on the gold-platinum-silicon-based nanoenzyme prepared in the step 4 in the example 1, and the result of mapping analysis on gold, platinum, silicon, mercury and oxygen elements is shown in figure 8, so that the result shows that on the basis of successful preparation of the nanoenzyme, mercury ions interact with the nanoenzyme to be loaded on the surface of the nanoenzyme.
8. The ultraviolet absorbance analysis and color comparison of the reaction of the gold-platinum-silicon-based nanoenzyme prepared in step 4 of example 1 with mercury ions and sequence interfering ions show in fig. 9, and the results show that the prepared material has specificity for the detection of mercury ions, is not affected by other interfering ions, and has stable performance.
Application examples
The detection sensitivity of the nano enzyme prepared in the embodiment 1 to mercury ions is detected, and the detection method comprises the following steps:
(1) 200mmol/L acetic acid buffer solution with pH 5 and 60mmol/L H are prepared2O2100mmol/L TMB solution;
(2) preparation of 2.5X 10-10mol/L、2.5×10-9mol/L、2.5×10-8mol/L、2.5×10-7mol/L、2.5×10-6mol/L、2.5×10-5A mercury ion standard solution of mol/L;
(3) sequentially taking 10 mu L of acetic acid buffer solution and 6.67 mu L of H2O2The solution was put on an ELISA plate, and 5. mu.L of the nanoenzyme prepared in example 1 was added thereto to obtain solution X.
(4) Taking 2.5X 10 in the step (2)-10mol/L、2.5×10-9mol/L、2.5×10-8mol/L、2.5×10- 7mol/L、2.5×10-6mol/L、2.5×10-5mixing 10 μ L of mol/L mercury ion standard solution with the solution X in the step (3) to obtain a mixed solution 1, a mixed solution 2, a mixed solution 3, a mixed solution 4, a mixed solution 5 and a mixed solution 6, adding 0.4 μ L TMB solution into the mixed solution, and standing for 20min toThe color reaction was complete and the change in color was observed and recorded.
(5) And (5) carrying out ultraviolet absorbance detection analysis on the solution which is well stood in the step (4), recording the absorbance at 650nm and recording experimental data.
(6) And (3) linearly fitting according to the absorbance data in the step (5) and the corresponding mercury ion standard concentration to obtain a mercury ion content conversion formula y ═ kx + b, wherein y is the ultraviolet absorbance of the mixed solution 1, the mixed solution 2, the mixed solution 3, the mixed solution 4, the mixed solution 5 and the mixed solution 6, and x is the mercury ion concentration, wherein the k value is the slope, and b is a constant, and the result is shown in fig. 10.
From the above results, it was found that when the TMB solution was added to the mixed solution and left to stand for 20min, the color of the solution changed from dark blue to light blue, and even a small amount of mercury ions produced a significant colorimetric change and were easily distinguished by the naked eye. Spectrum of TMB system, mercury ion concentration from 2.5X 10-10The mol/L is increased to 2.5X 10-5At mol/L, the absorbance of the TMB system at 650nm gradually decreases. The absorbance at 650nm of the solution to mercury ions was 2.5X 10-10mol/L～2.5×10-5The correlation coefficient is 0.9659, and the detection limit (signal-to-noise ratio is 3) of mercury ions is 5 pmol/L. The nano enzyme has high sensitivity on the qualitative detection of the mercury ions, and simultaneously, the relationship between the concentration of the mercury ions and the absorbance is constructed, thereby providing great convenience for the later detection of the concentration of the mercury ions.
The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.
Claims (10)
1. A preparation method of nano enzyme is characterized by comprising the following steps:
taking a cationic surfactant as a template, adding a silicon source, a catalyst, a structure directing agent and an organic solvent, and reacting to obtain silicon spheres;
carrying out amination treatment on the silicon spheres to obtain silicon amide spheres;
dispersing the silicon amide spheres in water, adding the gold dispersion liquid, and stirring to obtain a silicon sphere-nanogold compound;
and dispersing the silicon sphere-nano gold compound in water, adding a platinum-containing solution and a sodium citrate solution, stirring, adding a sodium borohydride solution, and stirring for reaction to obtain the gold-platinum-silicon-based nano enzyme.
2. The process of preparing nanoenzyme according to claim 1, wherein the cationic surfactant is used as a template, and a silicon source, a catalyst and a structure directing agent are added to react to obtain silicon spheres, wherein the reaction comprises the following steps:
the cationic surfactant comprises cetyl trimethyl ammonium bromide; and/or the presence of a gas in the gas,
the silicon source comprises tetraethoxysilane; and/or the presence of a gas in the gas,
the catalyst comprises triethanolamine; and/or the presence of a gas in the gas,
the structure directing agent comprises sodium salicylate.
3. The method for preparing nanoenzyme according to claim 2, wherein the step of adding a silicon source, a catalyst and a structure directing agent into the cationic surfactant as a template to obtain the silica spheres through reaction comprises:
adding triethanolamine into water, and stirring at 80 deg.C for 30-60min to obtain solution A;
adding hexadecyl trimethyl ammonium bromide and sodium salicylate into the solution A, and stirring for 1 hour at constant temperature to obtain a solution B;
adding tetraethoxysilane into the solution B, stirring at constant temperature, and reacting for 12-15h to obtain a reaction solution C;
centrifuging and washing the reaction solution C to remove redundant reactants to obtain a precipitate, dispersing the precipitate in a mixed solution of hydrochloric acid and methanol, and stirring at 60 ℃ for 6-10h to obtain a solution D;
and centrifuging and washing the solution D to remove redundant reactants to obtain a precipitate, re-dispersing the obtained precipitate in ethanol, and placing the ethanol at a temperature lower than room temperature for later use to obtain the silicon spheres.
4. The method for preparing nanoenzyme according to claim 1, wherein the step of subjecting the silica spheres to amination treatment to obtain aminated silica spheres comprises:
adding (3-aminopropyl) trimethoxy silane into the silicon spheres, and stirring, centrifuging and washing to obtain the silicon amide spheres.
5. The method according to claim 4, wherein the volume ratio of the silicon source to the (3-aminopropyl) trimethoxysilane is 1: (24-28).
6. The process for preparing nanoenzyme according to claim 4, wherein the stirring time is 5-7 h; and/or the presence of a gas in the atmosphere,
the rotation speed of the centrifugation is 5000-; and/or the presence of a gas in the gas,
the centrifugation time is 4-10 min.
7. The method for preparing nanoenzyme according to claim 1, wherein in the step of dispersing the silicon amide spheres in water, adding the gold dispersion, and stirring to obtain the silicon sphere-nanogold composite, 2mL of silicon source is reacted to obtain silicon amide spheres, and (120- "122") nmoL gold is added.
8. The method of claim 1, wherein the step of dispersing the silicon sphere-nanogold complex in water, adding a platinum-containing solution and a sodium citrate solution, stirring, adding a sodium borohydride solution, and stirring to obtain the gold-platinum-silicon-based nanoenzyme comprises the step of reacting, wherein the platinum-containing solution comprises H2PtCl6An aqueous solution.
9. The method for preparing nanoenzyme according to claim 1, wherein in the step of dispersing the silicon sphere-nanogold complex in water, adding a platinum-containing solution and a sodium citrate solution, stirring, adding a sodium borohydride solution, and stirring to react to obtain the gold-platinum-silicon-based nanoenzyme, each 2mL of the silicon source is added with (8-9) mmoL of platinum correspondingly.
10. Use of a nanoenzyme prepared by the process of any one of claims 1 to 9 for the detection of mercury ions.
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| CN111744472A (en) * | 2020-06-19 | 2020-10-09 | 湖北工业大学 | Preparation method and application of porous silicon platinum nano enzyme material |
| CN112619666A (en) * | 2020-11-30 | 2021-04-09 | 深圳市人民医院 | Preparation method and application of central radial-diplopore nano composite material |
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