CN111020871A - Nanofiber membrane for enzyme immobilization and preparation method thereof - Google Patents
Nanofiber membrane for enzyme immobilization and preparation method thereof Download PDFInfo
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Abstract
The invention provides a nanofiber membrane for enzyme immobilization and a preparation method thereof, wherein the method comprises the following steps: (1) preparation of PEG-PMAH-PAA: preparing PEG-PMAH from maleic anhydride and polyethylene glycol, and preparing PEG-PMAH-PAA from PEG-PMAH and acrylic acid; (2) preparing an electrostatic spinning solution: preparing electrostatic spinning solution by taking PEG-PMAH-PAA and gold nanoparticles as raw materials; (3) preparing a nanofiber membrane: and spinning the electrostatic spinning solution by adopting an electrostatic spinning technology to obtain the nanofiber membrane. The addition of the gold nanoparticles in the nanofiber membrane improves the adsorption capacity of the enzyme on the nanofiber membrane, so that the nanofiber membrane has higher enzyme immobilization capacity, the structure of the enzyme is not influenced, and the original biological activity of the enzyme is maintained, so that the nanofiber membrane prepared by the method is suitable for being widely applied as an immobilization material of an enzyme solution.
Description
Technical Field
The invention belongs to the technical field of membrane materials, and particularly relates to a nanofiber membrane for enzyme immobilization and a preparation method thereof.
Background
The enzyme is a protein with a catalytic function, is used as a biocatalyst, and has the advantages of high reaction rate, mild reaction conditions, strong substrate specificity and the like. Enzymes have been widely used in many fields such as food, medicine, light industry and agriculture. However, enzymes have the following disadvantages: poor thermal stability, extreme sensitivity to acid and alkali, difficult separation of free enzyme after reaction with a substrate and a product, incapability of being reused and the like, thereby limiting the application of the enzyme. The immobilized enzyme technology overcomes the defects of the enzyme, thereby opening up a wide prospect for the application of the enzyme.
The carrier of the immobilized enzyme is a hot problem of research, and in recent years, the electrostatic spinning nanofiber is widely emphasized in the research of enzyme immobilization, has a large specific surface area and a high porosity, and can effectively improve the enzyme loading capacity and reduce the diffusion resistance of a substrate during enzyme catalysis. The existing principle of enzyme immobilization by electrostatic spinning nanofiber membranes is mainly to immobilize enzymes in an adsorption or covalent bond manner, the enzyme and the nanofibers are poor in firmness of electrostatic adsorption combination, and the firmness of enzyme combination can be improved to a certain extent by the covalent connection manner, but the structure of the enzyme is easily changed, so that the biological activity of the enzyme is influenced. Therefore, it is necessary to provide a nanofiber membrane having improved enzyme immobilization strength without changing the structure of the enzyme.
Disclosure of Invention
In order to solve the problems of the prior art, the present invention aims to provide a nanofiber membrane for enzyme immobilization and a preparation method thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of a nanofiber membrane for enzyme immobilization comprises the following steps:
(1) preparation of PEG-PMAH-PAA
Adding maleic anhydride and polyethylene glycol into a toluene solution according to a molar ratio of 1:1-2, stirring and mixing, adding sodium p-toluenesulfonate under the condition of stirring and under the protection of nitrogen, slowly heating to 110-120 ℃, and keeping the temperature for reacting for 3-5h to obtain an orange transparent solution; then extracting the obtained reaction liquid with ether, combining the extract liquid and drying the extract liquid to obtain PEG-PMAH;
mixing the obtained PEG-PMAH with acrylic acid in a molar ratio of 1:5-10, adding a toluene solution of dodecyl mercaptan, slowly heating to 65 ℃ under stirring, slowly dropwise adding a toluene solution of azobisisobutyronitrile, and keeping the temperature to continuously stir and react for 2-3 hours after dropwise adding to obtain a reaction product; performing rotary evaporation on the reaction product on a rotary evaporator to remove the toluene solution, extracting the residual solution with diethyl ether, combining the extract liquor and drying to obtain PEG-PMAH-PAA;
(2) preparation of electrospinning solution
Adding the PEG-PMAH-PAA prepared in the step (1) into a dimethylformamide solution to dissolve the PEG-PMAH-PAA, then adding gold nanoparticles, magnetically stirring for 2-3h, and then carrying out ultrasonic treatment for 20-40min to obtain an electrostatic spinning solution;
(3) preparation of nanofiber membranes
And (3) spinning the electrostatic spinning solution prepared in the step (2) by adopting an electrostatic spinning technology to obtain the nanofiber membrane.
Further, the specific conditions of the electrostatic spinning in the step (3) are as follows: the voltage is 20-40kV, the distance between the spinneret orifice and the receiving screen is 8-15cm, the inner diameter of the spinning hole is 0.5-1.0mm, and the spinning speed is 20-50 mu L/min.
Furthermore, the addition amount of the sodium p-toluenesulfonate is 1/50-1/100 of the molar mass of the maleic anhydride.
Further, the polyethylene glycol in the step (1) is polyethylene glycol 400, 600, 800 or 1000.
Further, the molar mass of the added dodecyl mercaptan in the step (1) is 1/30-1/50 of acrylic acid, and the molar mass of the added azobisisobutyronitrile is 1/50-1/80 of acrylic acid.
Further, the particle size of the gold nanoparticles in the step (2) is 15-20 nm.
The nanofiber membrane prepared by the preparation method is provided.
The method for carrying out enzyme immobilization by using the nanofiber membrane comprises the following steps: washing the nanofiber membrane, immersing the washed nanofiber membrane into 0.01M phosphate buffer solution with the pH value of 7.2, then adding enzyme solution, and carrying out shake reaction for 3-6h at room temperature; and taking out the nanofiber membrane, and then washing with deionized water for 2-3 times to obtain the nanofiber membrane immobilized with the enzyme.
Has the advantages that: the invention provides a nanofiber membrane for enzyme immobilization and a preparation method thereof, the nanofiber membrane prepared by the invention is prepared by an electrostatic spinning technology, gold nanoparticles are added into electrostatic spinning liquid, and the addition of the gold nanoparticles improves the adsorption capacity of enzyme on the nanofiber membrane, so that the nanofiber membrane has higher enzyme immobilization capacity, the structure of the enzyme is not influenced, and the original biological activity of the enzyme is maintained, therefore, the nanofiber membrane prepared by the invention is suitable for being widely applied as an immobilization material of an enzyme solution.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
In the following examples and comparative examples, papain was used as an enzyme solution, and the amount of the enzyme solution added was 1mg, and gold nanoparticles used were a commercially available gold nanoparticle solution having a concentration of 1M.
The enzyme loading was measured as follows:
the Bradford method was used to determine the immobilization of the enzyme on the nanofiber membrane. The protein content is determined by a Coomassie brilliant blue method, the absorbance of the protein solution is detected at 595nm by an ultraviolet spectrophotometer, the protein content in the enzyme solution before and after immobilization and PBS used for washing the immobilized enzyme membrane is tested, and the protein concentration is determined by comparing with a BSA standard curve. The enzyme loading was calculated according to the following formula.
In the formula, AeEnzyme loading (mg/g membrane); c0The protein concentration (mg/mL) in the enzyme solution before immobilization; c is the protein concentration (mg/mL) in the immobilized enzyme solution; v is the volume (mL) of the enzyme solution used for immobilizing the enzyme; cWProtein concentration (mg/mL) in PBS buffer after washing of immobilized enzyme; vWVolume of PBS buffer used (mL); w is the mass of the nanofiber membrane used.
Example 1
A preparation method of a nanofiber membrane for enzyme immobilization comprises the following steps:
(1) preparation of PEG-PMAH-PAA
Adding maleic anhydride and polyethylene glycol into a toluene solution according to a molar ratio of 1:1.5, stirring and mixing, adding sodium p-toluenesulfonate under the condition of stirring and under the protection of nitrogen, slowly heating to 115 ℃, and keeping the temperature for reacting for 4 hours to obtain an orange transparent solution; then extracting the obtained reaction liquid with ether, combining the extract liquid and drying the extract liquid to obtain PEG-PMAH;
mixing the PEG-PMAH obtained above with acrylic acid in a molar ratio of 1:8, adding 70mL of a toluene solution of dodecyl mercaptan, slowly heating to 65 ℃ under stirring, slowly dropwise adding the toluene solution of azobisisobutyronitrile, and keeping the temperature to continuously stir and react for 2.5 hours after dropwise adding to obtain a reaction product; performing rotary evaporation on the reaction product on a rotary evaporator to remove the toluene solution, extracting the residual solution with diethyl ether, combining the extract liquor and drying to obtain PEG-PMAH-PAA;
(2) preparation of electrospinning solution
Adding the PEG-PMAH-PAA prepared in the step (1) into a dimethylformamide solution to dissolve the PEG-PMAH-PAA, then adding a gold nanoparticle solution with the final concentration of 150nM, magnetically stirring for 2.5h, and then carrying out ultrasonic treatment for 30min to obtain an electrostatic spinning solution; the particle size of the gold nanoparticles is 15-20 nm;
(3) preparation of nanofiber membranes
And (3) spinning the electrostatic spinning solution prepared in the step (2) by adopting an electrostatic spinning technology to obtain the nanofiber membrane.
The specific conditions of the electrostatic spinning in the step (3) are as follows: the voltage is 30kV, the distance between a spinning nozzle and a receiving screen is 12cm, the inner diameter of a spinning hole is 0.8mm, and the spinning speed is 35 mu L/min.
The addition amount of the sodium p-toluenesulfonate is 1/80 of the molar mass of the maleic anhydride.
The polyethylene glycol in the step (1) is polyethylene glycol 600.
The molar mass of the added dodecyl mercaptan in the step (1) is 1/40 of acrylic acid, and the molar mass of the added azobisisobutyronitrile is 1/70 of acrylic acid.
The method for enzyme immobilization of the nanofiber membrane described in this embodiment includes the following steps: washing the nanofiber membrane, immersing the washed nanofiber membrane into 0.01M phosphate buffer solution with the pH value of 7.2, then adding enzyme solution, and carrying out shake reaction for 4.5 hours at room temperature; and taking out the nanofiber membrane, and then washing with deionized water for 2-3 times to obtain the nanofiber membrane immobilized with the enzyme. The enzyme loading was 186 mg/g.
Example 2
A preparation method of a nanofiber membrane for enzyme immobilization comprises the following steps:
(1) preparation of PEG-PMAH-PAA
Adding maleic anhydride and polyethylene glycol into a toluene solution according to a molar ratio of 1:1, stirring and mixing, adding sodium p-toluenesulfonate under the condition of stirring and under the protection of nitrogen, slowly heating to 110 ℃, and keeping the temperature for reacting for 3 hours to obtain an orange transparent solution; then extracting the obtained reaction liquid with ether, combining the extract liquid and drying the extract liquid to obtain PEG-PMAH;
mixing the obtained PEG-PMAH with acrylic acid in a molar ratio of 1:5, adding a toluene solution of dodecyl mercaptan, slowly heating to 65 ℃ under the condition of stirring, slowly dropwise adding a toluene solution of azobisisobutyronitrile, and keeping the temperature to continuously stir and react for 2 hours after dropwise adding to obtain a reaction product; performing rotary evaporation on the reaction product on a rotary evaporator to remove the toluene solution, extracting the residual solution with diethyl ether, combining the extract liquor and drying to obtain PEG-PMAH-PAA;
(2) preparation of electrospinning solution
Adding the PEG-PMAH-PAA prepared in the step (1) into a dimethylformamide solution to dissolve the PEG-PMAH-PAA, then adding gold nanoparticles with the final concentration of 50nM, magnetically stirring for 2h, and then carrying out ultrasonic treatment for 20min to obtain an electrostatic spinning solution; the particle size of the gold nanoparticles is 15-20 nm;
(3) preparation of nanofiber membranes
And (3) spinning the electrostatic spinning solution prepared in the step (2) by adopting an electrostatic spinning technology to obtain the nanofiber membrane.
The specific conditions of the electrostatic spinning in the step (3) are as follows: the voltage is 20kV, the distance between a spinning nozzle and a receiving screen is 8cm, the inner diameter of a spinning hole is 0.5mm, and the spinning speed is 20 mu L/min.
The addition amount of the sodium p-toluenesulfonate is 1/50 of the molar mass of the maleic anhydride.
The polyethylene glycol in the step (1) is polyethylene glycol 400.
The molar mass of the added dodecyl mercaptan in the step (1) is 1/30 of acrylic acid, and the molar mass of the added azobisisobutyronitrile is 1/50 of acrylic acid.
The method for enzyme immobilization of the nanofiber membrane described in this embodiment includes the following steps: washing the nanofiber membrane, immersing the washed nanofiber membrane into 0.01M phosphate buffer solution with the pH value of 7.2, then adding enzyme solution, and carrying out shake reaction for 3 hours at room temperature; and taking out the nanofiber membrane, and then washing with deionized water for 2-3 times to obtain the nanofiber membrane immobilized with the enzyme. The enzyme loading was 138 mg/g.
Example 3
A preparation method of a nanofiber membrane for enzyme immobilization comprises the following steps:
(1) preparation of PEG-PMAH-PAA
Adding maleic anhydride and polyethylene glycol into a toluene solution according to a molar ratio of 1:1.2, stirring and mixing, adding sodium p-toluenesulfonate under the condition of stirring and under the protection of nitrogen, slowly heating to 112 ℃, and keeping the temperature to react for 3.5 hours to obtain an orange transparent solution; then extracting the obtained reaction liquid with ether, combining the extract liquid and drying the extract liquid to obtain PEG-PMAH;
mixing the PEG-PMAH obtained above with acrylic acid in a molar ratio of 1:6, adding a toluene solution of dodecyl mercaptan, slowly heating to 65 ℃ under the condition of stirring, slowly dropwise adding a toluene solution of azobisisobutyronitrile, and keeping the temperature to continuously stir and react for 2.3 hours after dropwise adding to obtain a reaction product; performing rotary evaporation on the reaction product on a rotary evaporator to remove the toluene solution, extracting the residual solution with diethyl ether, combining the extract liquor and drying to obtain PEG-PMAH-PAA;
(2) preparation of electrospinning solution
Adding the PEG-PMAH-PAA prepared in the step (1) into a dimethylformamide solution to dissolve the PEG-PMAH-PAA, then adding gold nanoparticles with the final concentration of 100nM, magnetically stirring for 2.2h, and then carrying out ultrasonic treatment for 25min to obtain an electrostatic spinning solution; the particle size of the gold nanoparticles is 15-20 nm;
(3) preparation of nanofiber membranes
And (3) spinning the electrostatic spinning solution prepared in the step (2) by adopting an electrostatic spinning technology to obtain the nanofiber membrane.
The specific conditions of the electrostatic spinning in the step (3) are as follows: the voltage is 25kV, the distance between a spinning nozzle and a receiving screen is 10cm, the inner diameter of a spinning hole is 0.6mm, and the spinning speed is 30 mu L/min.
The addition amount of the sodium p-toluenesulfonate is 1/70 of the molar mass of the maleic anhydride.
The polyethylene glycol in the step (1) is polyethylene glycol 800.
The molar mass of the added dodecyl mercaptan in the step (1) is 1/35 of acrylic acid, and the molar mass of the added azobisisobutyronitrile is 1/60 of acrylic acid.
The method for enzyme immobilization of the nanofiber membrane described in this embodiment includes the following steps: washing the nanofiber membrane, immersing the washed nanofiber membrane into 0.01M phosphate buffer solution with the pH value of 7.2, then adding enzyme solution, and carrying out shake reaction for 4 hours at room temperature; and taking out the nanofiber membrane, and then washing with deionized water for 2-3 times to obtain the nanofiber membrane immobilized with the enzyme. The enzyme loading was 154 mg/g.
Example 4
A preparation method of a nanofiber membrane for enzyme immobilization comprises the following steps:
(1) preparation of PEG-PMAH-PAA
Adding maleic anhydride and polyethylene glycol into a toluene solution according to a molar ratio of 1:2, stirring and mixing, adding sodium p-toluenesulfonate under the condition of stirring and under the protection of nitrogen, slowly heating to 120 ℃, and keeping the temperature to react for 5 hours to obtain an orange transparent solution; then extracting the obtained reaction liquid with ether, combining the extract liquid and drying the extract liquid to obtain PEG-PMAH;
mixing the obtained PEG-PMAH with acrylic acid in a molar ratio of 1:10, adding a toluene solution of dodecyl mercaptan, slowly heating to 65 ℃ under the condition of stirring, slowly dropwise adding a toluene solution of azobisisobutyronitrile, and keeping the temperature to continuously stir and react for 3 hours after dropwise adding to obtain a reaction product; performing rotary evaporation on the reaction product on a rotary evaporator to remove the toluene solution, extracting the residual solution with diethyl ether, combining the extract liquor and drying to obtain PEG-PMAH-PAA;
(2) preparation of electrospinning solution
Adding the PEG-PMAH-PAA prepared in the step (1) into a dimethylformamide solution to dissolve the PEG-PMAH-PAA, then adding gold nanoparticles with the final concentration of 200nM, magnetically stirring for 3h, and then carrying out ultrasonic treatment for 40min to obtain an electrostatic spinning solution; the particle size of the gold nanoparticles is 15-20 nm;
(3) preparation of nanofiber membranes
And (3) spinning the electrostatic spinning solution prepared in the step (2) by adopting an electrostatic spinning technology to obtain the nanofiber membrane.
The specific conditions of the electrostatic spinning in the step (3) are as follows: the voltage is 40kV, the distance between a spinning nozzle and a receiving screen is 15cm, the inner diameter of a spinning hole is 1.0mm, and the spinning speed is 50 mu L/min.
The addition amount of the sodium p-toluenesulfonate is 1/100 of the molar mass of the maleic anhydride.
The polyethylene glycol in the step (1) is polyethylene glycol 1000.
The molar mass of the added dodecyl mercaptan in the step (1) is 1/50 of acrylic acid, and the molar mass of the added azobisisobutyronitrile is 1/80 of acrylic acid.
The method for enzyme immobilization of the nanofiber membrane described in this embodiment includes the following steps: washing the nanofiber membrane, immersing the washed nanofiber membrane into 0.01M phosphate buffer solution with the pH value of 7.2, then adding enzyme solution, and carrying out shake reaction for 6 hours at room temperature; and taking out the nanofiber membrane, and then washing with deionized water for 2-3 times to obtain the nanofiber membrane immobilized with the enzyme. The enzyme loading was 167 mg/g.
Comparative example 1
Comparative example 1 is different from example 1 in that comparative example 1 lacks step (2), and the nanofiber membrane obtained therefrom has an enzyme loading of 65 mg/g.
The test results of the above examples and comparative examples show that the nanofiber membrane prepared by the invention has higher enzyme immobilization amount, and the comparative examples show that the nanofiber membrane doped with gold nanoparticles plays an important role in improving the enzyme immobilization amount, so that the nanofiber membrane prepared by the invention is suitable for being widely applied as an immobilization material of an enzyme solution.
Claims (8)
1. A preparation method of a nanofiber membrane for enzyme immobilization is characterized by comprising the following steps:
(1) preparation of PEG-PMAH-PAA
Adding maleic anhydride and polyethylene glycol into a toluene solution according to a molar ratio of 1:1-2, stirring and mixing, adding sodium p-toluenesulfonate under the condition of stirring and under the protection of nitrogen, slowly heating to 110-120 ℃, and keeping the temperature for reacting for 3-5h to obtain an orange transparent solution; then extracting the obtained reaction liquid with ether, combining the extract liquid and drying the extract liquid to obtain PEG-PMAH;
mixing the obtained PEG-PMAH with acrylic acid in a molar ratio of 1:5-10, adding a toluene solution of dodecyl mercaptan, slowly heating to 65 ℃ under stirring, slowly dropwise adding a toluene solution of azobisisobutyronitrile, and keeping the temperature to continuously stir and react for 2-3 hours after dropwise adding to obtain a reaction product; performing rotary evaporation on the reaction product on a rotary evaporator to remove the toluene solution, extracting the residual solution with diethyl ether, combining the extract liquor and drying to obtain PEG-PMAH-PAA;
(2) preparation of electrospinning solution
Adding the PEG-PMAH-PAA prepared in the step (1) into a dimethylformamide solution to dissolve the PEG-PMAH-PAA, then adding gold nanoparticles, magnetically stirring for 2-3h, and then carrying out ultrasonic treatment for 20-40min to obtain an electrostatic spinning solution;
(3) preparation of nanofiber membranes
And (3) spinning the electrostatic spinning solution prepared in the step (2) by adopting an electrostatic spinning technology to obtain the nanofiber membrane.
2. The method for preparing nanofiber membrane for enzyme immobilization as claimed in claim 1, wherein the specific conditions of electrospinning in step (3) are as follows: the voltage is 20-40kV, the distance between the spinneret orifice and the receiving screen is 8-15cm, the inner diameter of the spinning hole is 0.5-1.0mm, and the spinning speed is 20-50 mu L/min.
3. The method of claim 1, wherein the sodium p-toluenesulfonate is added in an amount of 1/50-1/100 based on the molar mass of maleic anhydride.
4. The method of claim 1, wherein the polyethylene glycol in step (1) is polyethylene glycol 400, 600, 800 or 1000.
5. The method of claim 1, wherein the molar mass of the dodecyl mercaptan added in step (1) is 1/30-1/50 and the molar mass of the azobisisobutyronitrile added is 1/50-1/80.
6. The method for preparing nanofiber membrane for enzyme immobilization as claimed in claim 1, wherein the gold nanoparticles in step (2) have a particle size of 15-20 nm.
7. The nanofiber membrane produced by the production method as set forth in any one of claims 1 to 6.
8. A method for enzyme immobilization using the nanofiber membrane as claimed in claim 7, comprising the steps of: washing the nanofiber membrane, immersing the washed nanofiber membrane into 0.01M phosphate buffer solution with the pH value of 7.2, then adding enzyme solution, and carrying out shake reaction for 3-6h at room temperature; and taking out the nanofiber membrane, and then washing with deionized water for 2-3 times to obtain the nanofiber membrane immobilized with the enzyme.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114806179A (en) * | 2022-03-31 | 2022-07-29 | 宁波东致杰电力科技有限公司 | Insulating liquid silicone rubber and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737560A (en) * | 2005-09-02 | 2006-02-22 | 天津大学 | Static electricity spinning prepared fixed enzymic electrode and method thereof |
| CN101290301A (en) * | 2008-05-23 | 2008-10-22 | 浙江大学 | Ampere enzyme electrode based on electrostatic spinning fiber membrane and method for making same |
| CN101915797A (en) * | 2010-07-27 | 2010-12-15 | 北京师范大学 | Method for preparing electrostatic spinning immobilized laccase electrode |
| CN104593892A (en) * | 2015-01-25 | 2015-05-06 | 北京化工大学 | Preparation method for nanogold-enhanced fluorescence sheath-core structure nano fiber |
| CN104888215A (en) * | 2014-03-07 | 2015-09-09 | 中国科学院化学研究所 | Fiber film coated with gold nanorods, and preparation method and application thereof |
| CN110292652A (en) * | 2018-03-23 | 2019-10-01 | 国家纳米科学中心 | Mercaptophenyl boronic acid activates gold nano grain, preparation method and application |
-
2019
- 2019-12-31 CN CN201911412036.XA patent/CN111020871A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1737560A (en) * | 2005-09-02 | 2006-02-22 | 天津大学 | Static electricity spinning prepared fixed enzymic electrode and method thereof |
| CN101290301A (en) * | 2008-05-23 | 2008-10-22 | 浙江大学 | Ampere enzyme electrode based on electrostatic spinning fiber membrane and method for making same |
| CN101915797A (en) * | 2010-07-27 | 2010-12-15 | 北京师范大学 | Method for preparing electrostatic spinning immobilized laccase electrode |
| CN104888215A (en) * | 2014-03-07 | 2015-09-09 | 中国科学院化学研究所 | Fiber film coated with gold nanorods, and preparation method and application thereof |
| CN104593892A (en) * | 2015-01-25 | 2015-05-06 | 北京化工大学 | Preparation method for nanogold-enhanced fluorescence sheath-core structure nano fiber |
| CN110292652A (en) * | 2018-03-23 | 2019-10-01 | 国家纳米科学中心 | Mercaptophenyl boronic acid activates gold nano grain, preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| 胡伟兆等: "大分子分散改性剂PEG400-PMAH-PAA的合成及应用", 《合成化学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114806179A (en) * | 2022-03-31 | 2022-07-29 | 宁波东致杰电力科技有限公司 | Insulating liquid silicone rubber and preparation method and application thereof |
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