CN109337811B - Eutectic solvent monolithic column enzyme reactor and preparation method thereof - Google Patents
Eutectic solvent monolithic column enzyme reactor and preparation method thereof Download PDFInfo
- Publication number
- CN109337811B CN109337811B CN201811195644.5A CN201811195644A CN109337811B CN 109337811 B CN109337811 B CN 109337811B CN 201811195644 A CN201811195644 A CN 201811195644A CN 109337811 B CN109337811 B CN 109337811B
- Authority
- CN
- China
- Prior art keywords
- enzyme
- eutectic solvent
- monolithic column
- capillary
- column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/04—Acids; Metal salts or ammonium salts thereof
- C08F220/06—Acrylic acid; Methacrylic acid; Metal salts or ammonium salts thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/12—Esters of monohydric alcohols or phenols
- C08F220/14—Methyl esters, e.g. methyl (meth)acrylate
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
- C08F222/10—Esters
- C08F222/1006—Esters of polyhydric alcohols or polyhydric phenols
- C08F222/102—Esters of polyhydric alcohols or polyhydric phenols of dialcohols, e.g. ethylene glycol di(meth)acrylate or 1,4-butanediol dimethacrylate
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
- C08F222/36—Amides or imides
- C08F222/38—Amides
- C08F222/385—Monomers containing two or more (meth)acrylamide groups, e.g. N,N'-methylenebisacrylamide
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Polymers & Plastics (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Sustainable Development (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a eutectic solvent monolithic column enzyme reactor and a preparation method thereof. The preparation method comprises the following steps: adding a free radical initiator azobisisobutyronitrile into a eutectic solvent/ionic liquid, performing ultrasonic dispersion, adding a functional monomer methacrylic acid, a comonomer butyl methacrylate and a crosslinking agent ethylene glycol dimethacrylate, performing ultrasonic dissolution, injecting into a capillary, performing thermal initiation synthesis on an integral column, reducing a disulfide bond in trypsin to a sulfhydryl group by using a tris (2-carbonyl ethyl) phosphate (TCEP-HCl) reagent, adding ammonium persulfate and N, N' -methylene bisacrylamide to prepare an enzyme derivative solution, reacting with the integral column at room temperature, and fixing the enzyme derivative solution on the surface of the integral column to perform enzymolysis on protein. The monolithic column has stable structure and low influence on enzyme activity, and can be used as a novel enzyme reactor enzymolysis protein solution for recycling. The invention adopts the in-situ polymerization method to directly polymerize in the capillary, and the synthesis method is simple and easy to implement, simple to operate, short in polymerization time, low in cost and wide in application prospect.
Description
Technical Field
The invention relates to a eutectic solvent monolithic column enzyme reactor and a preparation method thereof, namely a capillary monolithic column enzyme reactor prepared by taking the eutectic solvent as a green pore-foaming agent and application thereof in enzymolysis of protein.
Background
Proteomics is a science for studying proteome by scientific and technical means, and analyzes dynamically-changed proteins in cells from the whole. The traditional method for carrying out enzymolysis on protein by using solution has the problems of long enzymolysis time, low analysis flux and self-hydrolysis, and is difficult to be used for analyzing trace protein samples. At present, the main enzyme reactors mainly comprise a monolithic enzyme reactor, a particle immobilized enzyme reactor and an open-tube enzyme reactor, wherein the monolithic enzyme reactor is widely applied. Monolithic columns are continuous monolithic porous materials formed by in situ polymerization reactions in channels within the column. The types of the monolith enzyme reactors can be classified into organic monolith enzyme reactors and inorganic monolith enzyme reactors according to the difference of monomers in the monolith enzyme reactors. Since the organic monolithic column enzyme reactor uses chemical reaction to fix enzyme, the space conformation change of the enzyme can be caused in the curing process, the enzyme activity is easy to be reduced, and the inorganic monolithic column enzyme reactor uses silicon dioxide as a monolithic material, compared with an organic monolithic column, the preparation is not easy, and the subsequent surface derivatization step is more complicated.
The choline eutectic solvent is a eutectic solvent formed by mixing quaternary ammonium salt (choline chloride) and polyalcohol (ethylene glycol) in a certain stoichiometric ratio. The eutectic solvent is essentially an ionic mixture formed by mixing a plurality of compounds with different melting points according to a certain proportion, the physical and chemical properties of the eutectic solvent are very similar to those of ionic liquid, and the eutectic solvent is also called as novel ionic liquid. The eutectic solvent has the advantages of low toxicity, degradability, simple preparation and low price, and the atom utilization rate in the synthesis process reaches 100%. Compared with the traditional pore-foaming agents such as toluene and the like, the organic silicon-carbon composite material is an ideal green solvent.
Click chemistry refers to the formation of highly selective carbon-heteroatom bonds by the splicing of small units, with simple reaction conditions, environmental friendliness, and strong stereoselectivity. The 'mercapto-alkene' click reaction takes photo-initiated free radical reaction as a catalyst, and has high selectivity in reaction between a specific region and a functional group. The click reaction fixes the enzyme on the monolithic column by utilizing the reaction of residual double bonds on the monolithic column and sulfydryl of trypsin, is an effective enzyme fixing method, and can avoid the influence on permeability caused by secondary grafting on the monolithic column.
Disclosure of Invention
The invention aims to provide a eutectic solvent monolithic column enzyme reactor and a preparation method thereof. The eutectic solvent is used as a green pore-foaming agent, so that the permeability of the whole column is improved, the flux of a sample liquid is increased, the enzyme activity is retained to the maximum extent, and the enzymolysis efficiency is ensured. Preparing a capillary monolithic column by an in-situ polymerization method, and fixing trypsin on the surface of the monolithic column by applying a sulfydryl-alkene click reaction principle to obtain the monolithic column enzyme reactor. The invention adopts the in-situ polymerization method to directly polymerize in the capillary, and the synthesis method is simple and easy to implement, simple to operate, short in polymerization time and low in cost. The monolithic column has stable structure and low influence on enzyme activity, can be used as a novel enzyme reactor enzymolysis protein solution which is recycled, and has wide application prospect.
The invention provides a eutectic solvent monolithic column enzyme reactor which is prepared according to the following specific method steps:
1) adding choline chloride into ethylene glycol, heating in an oil bath for 4 hours, and completely dissolving to obtain a choline chloride-ethylene glycol eutectic solvent; wherein the mass ratio of the choline chloride to the ethylene glycol is 1: 3.
2) Dissolving Azodiisobutyronitrile (AIBN) serving as a free radical initiator into the eutectic solvent obtained in the step 1) and the ionic liquid, ultrasonically mixing the mixture, adding methacrylic acid (MAA), methyl methacrylate (BMA) and ethylene glycol dimethacrylate (EDMA) to obtain a pre-polymerized liquid, ultrasonically introducing nitrogen to remove oxygen dissolved in the solution. Injecting into capillary column pretreated with gamma-methacryloxypropyltrimethoxysilane (gamma-MPS), performing thermal polymerization at 65 deg.C for 1.5 hr, and removing unreacted reagent by flushing capillary column with acetonitrile.
Wherein the eutectic solvent and the ionic liquid are porogens of the monolithic column.
The ionic liquid is 1-hexyl-3-methylimidazole tetrafluoroborate; the mass ratio of the ionic liquid to the eutectic solvent is 3: 2; the molar ratio of the methacrylic acid to the methyl methacrylate to the ethylene glycol dimethacrylate is 48:163: 29; the inner diameter of the quartz capillary tube was 100. mu.m.
3) Reduction of disulfide bonds in trypsin: preparing 10 mg/mL trypsin solution and 1 mg/mL Tris (2-carbonylethyl) phosphate solution by using Tris (hydroxymethyl) aminomethane (Tris-HCl) buffer solution with the pH value of 8.0, reacting for 3 hours at 25 ℃ according to the volume ratio of the trypsin solution to the Tris (2-carbonylethyl) phosphate solution being 1:10, centrifuging for 10 minutes at 4 ℃, rotating at 10000 rpm, and removing insoluble substances; and after the solution is returned to the room temperature, 5 mL of supernatant is taken and added with 5 mg of ammonium persulfate and 10 mg of N, N' -methylene-bisacrylamide, and the mixture is shaken to be fully dissolved to obtain the enzyme derivative solution.
4) Injecting the enzyme derivative solution into the monolithic column in the step 2) by using a 1 mL syringe, reacting at room temperature for 5 hours to fix the enzyme on the surface of the monolithic column, washing with a Tris-HCl buffer solution with the pH of 8.0 for 1 hour after the reaction is finished to wash away unreacted substances, and storing in a refrigerator at the temperature of 4 ℃.
Wherein the volume ratio of the trypsin solution to the tris (2-carbonylethyl) phosphate solution is 10: 1;
blowing nitrogen for drying before the monolithic column in the step 4) reacts;
the concentrations of the ammonium persulfate and the N, N' -methylene-bisacrylamide are respectively 1 mg/mL and 2 mg/mL.
The eutectic solvent monolithic column enzyme reactor provided by the invention is used for fixing trypsin to carry out enzymolysis on protein, so that high flux of enzymolysis is realized.
The eutectic solvent monolithic column enzyme reactor provided by the invention has the following advantages:
1. the eutectic solvent is used as a green pore-foaming agent, is degradable and low in toxicity, can provide a good pore structure and permeability for the monolithic column, and has low influence on enzyme activity. The pore diameter can be adjusted by adjusting the ratio of the pore-foaming agent, so that the method is suitable for different reaction conditions.
2. The enzyme reactor synthesized by the method has high enzymolysis efficiency, can complete hydrolysis after 3 mM N-benzoyl-L-arginine ethyl ester is subjected to online enzymolysis for 15 s, can be repeatedly utilized, has good stability, greatly reduces enzymolysis time compared with the traditional solution enzymolysis method, and improves the enzymolysis efficiency.
3. The invention adopts the in-situ polymerization method to directly polymerize in the capillary, and the synthesis method is simple and easy to implement, simple to operate, short in polymerization time, low in cost and wide in application prospect.
Drawings
FIG. 1 is an infrared spectrum of a monolithic column.
FIG. 2 is a capillary electrophoresis chart of a sample of 2 mM N-benzoyl-L-arginine ethyl ester hydrolyzed by the enzyme reactor of the present invention.
FIG. 3 is a capillary electrophoresis chart of an enzyme reactor on-line enzymolysis 3 mM N-benzoyl-L-arginine ethyl ester sample.
Detailed Description
The present invention will be described in further detail with reference to the following examples. The experimental methods in the examples, in which specific conditions are not specified, are generally performed under the conditions described in the manual and the conventional conditions, or under the conditions recommended by the manufacturer; general equipment, materials, reagents and the like used are commercially available unless otherwise specified.
Example 1 preparation of eutectic solvent monolithic enzyme reactor
(1) Preparation of eutectic solvent monolithic column
1) Preparation of capillary monolithic column
Adding a free radical initiator azobisisobutyronitrile (2.0 mg), adding a eutectic solvent (80 mu L) and an ionic liquid (294 mu L), dispersing by using ultrasound for 5 min (650W), adding a functional monomer methacrylic acid (8.2 mu L, 0.096 mmol), a comonomer methyl methacrylate (46 mu L, 0.326 mmol) and a crosslinking monomer ethylene glycol dimethacrylate (18 mu L, 0.058 mmol), and completely dissolving by using ultrasound for 15 min. Nitrogen was introduced for 10 min, and the pre-polymerization solution was injected into the capillary with a syringe, sealed at both ends, and reacted at 53 ℃ for 1.5 h. After the reaction is finished, acetonitrile is used for washing away the residual pore-foaming agent ionic liquid, choline chloride-ethylene glycol and soluble substances in the monolithic column, and nitrogen is dried for later use.
2) Preparation of eutectic solvent monolithic column enzyme reactor (fixation of Trypsin)
A trypsin solution (10 mg/mL) and a Tris (2-carbonylethyl) phosphate solution (1 mg/mL) were prepared in a volume ratio of 1:10 using 20 mM Tris-HCl buffer (pH 8.0), reacted at 25 ℃ for 3 hours, and then centrifuged at 4 ℃ for 10 min (10000 rpm) to remove insoluble matter. Taking the supernatant, adding ammonium persulfate (1 mg/mL) and N, N' -methylene-bisacrylamide (2 mg/mL) to obtain the enzyme derivative solution.
The enzyme-derived solution was injected into the capillary monolith synthesized in 1), reacted at 25 ℃ for 5 hours, and after completion of the reaction, the column was washed with 20 mM Tris-HCl buffer (pH 8.0) for 1 hour to remove unreacted materials. The resulting monolith was stored at 4 ℃ for further use.
Example 2 measurement of enzyme Activity in eutectic solvent monolithic column enzyme reactor
In order to quantitatively obtain the enzyme activity of the eutectic solvent monolithic enzyme reactor, the performance of the enzyme reactor is characterized by a Km constant and a maximum enzyme reaction rate Vmax.
Michaelis constant (K)m) And maximum reaction velocity (V)max) Calculated from the michaelis equation. 1/V vs 1/[ S ] using Linewaver-Burk mapping]Plotting:
a straight line can be obtained. The intercept of the straight line on the horizontal axis is-1/Km, and the vertical intercept is 1/VmaxCan find KmAnd Vmax。
The above-described method (example 1) was used to synthesize a eutectic solvent monolithic column enzyme reactor, using N-benzoyl-L-arginine ethyl ester as the substrate for the enzyme reaction, and the substrate was hydrolyzed to benzoyl ester-L-arginine by trypsin, which has UV absorption at 214 nm. N-benzoyl-L-arginine ethyl ester was dissolved in 20 mM Tris-HCl buffer (pH 8.0) to prepare solutions (0.1 mM, 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 5 mM, 6 mM) at different concentrations, the substrate was injected into a capillary enzyme reactor using a syringe pump, the effluent was collected, and the peak area of the product benzoyl ester-L-arginine was measured using a high performance capillary electrophoresis apparatus (Agilent CE 7100).
The effective length of the eutectic solvent enzyme reactor is 5 cm, an empty capillary with the length of 50 cm and the inner diameter of 100 mu m is used for high-efficiency capillary electrophoresis analysis, the mobile phase is 20 mM Tris-HCl buffer solution (pH 8.0), the separation voltage is 15 kV, and the detection wavelength is 214 nm.
FIG. 1 is an infrared spectrum of a monolithic column.
FIG. 2 is a capillary electrophoresis chart of a sample hydrolyzed by 2 mM N-benzoyl-L-arginine ethyl ester in an enzyme reactor, and the separation effect of the product N-benzoyl-L-arginine and the unreacted substrate N-benzoyl-L-arginine ethyl ester in a capillary is good.
FIG. 3 is a capillary electrophoresis chart of an enzyme reactor on-line enzymolysis 3 mM N-benzoyl-L-arginine ethyl ester sample, and the hydrolysis can be completed within 15 s.
Claims (1)
1. A method for applying a eutectic solvent monolithic column enzyme reactor to enzymolysis of N-benzoyl-L-arginine ethyl ester is characterized by comprising the following steps:
preparation of mono-eutectic solvent monolithic column
1) Preparation of capillary monolithic column
Adding 2.0 mg of free radical initiator azobisisobutyronitrile into 80 mu L of eutectic solvent and 294 mu L of ionic liquid, dispersing for 5 min by ultrasonic, adding 8.2 mu L of functional monomer methacrylic acid of 0.096 mmol, 46 mu L of comonomer methyl methacrylate of 0.326 mmol, 18 mu L of crosslinking monomer ethylene glycol dimethacrylate of 0.058 mmol, and completely dissolving by ultrasonic for 15 min; introducing nitrogen for 10 min, injecting the pre-polymerization solution into the capillary tube by using an injector, sealing two ends, and reacting for 1.5 h at 53 ℃; after the reaction is finished, acetonitrile is used for washing away the residual pore-foaming agent ionic liquid, choline chloride-ethylene glycol and soluble substances in the monolithic column, and nitrogen is dried for later use;
2) preparation of eutectic solvent monolithic column enzyme reactor
Preparing 10 mg/mL trypsin solution and 1 mg/mL Tris (2-carbonylethyl) phosphate solution by using 20 mM Tris-HCl buffer solution with the pH value of 8.0, mixing the two solutions according to the volume ratio of 1:10, reacting for 3 h at 25 ℃, and then centrifuging for 10 min at 4 ℃ and at 10000 rpm to remove insoluble substances; taking the supernatant, adding 1 mg/mL ammonium persulfate and 2 mg/mL N, N' -methylene-bisacrylamide to obtain enzyme derived liquid;
injecting the enzyme derivative solution into the capillary monolithic column synthesized in 1), reacting at 25 ℃ for 5 h, and washing the column with 20 mM Tris-HCl buffer solution with pH 8.0 for 1 h after the reaction is completed to remove unreacted substances; storing the prepared monolithic column at 4 ℃ for later use;
enzyme activity determination of eutectic solvent monolithic column enzyme reactor
To quantitatively obtain the enzyme activity of the eutectic solvent monolithic enzyme reactor, the performance of the enzyme reactor was characterized by the Km constant and the maximum enzyme reaction rate Vmax:
michaelis constant (K)m) And maximum reaction velocity (V)max) Calculated from the Mie' S equation, 1/V vs. 1/[ S ] using the Linewaver-Burk mapping method]Plotting:
obtaining a straight line, wherein the intercept of the straight line on the horizontal axis is-1/Km, and the longitudinal intercept is 1/VmaxFinding KmAnd Vmax；
The reactor is used for synthesizing the eutectic solvent monolithic column enzyme, N-benzoyl-L-arginine ethyl ester is used as an enzyme reaction substrate, the substrate is hydrolyzed into benzoyl ester-L-arginine under the action of trypsin, and ultraviolet absorption is realized at a position of 214 nm; dissolving N-benzoyl-L-arginine ethyl ester in 20 mM Tris-HCl buffer solution with the pH value of 8.0 to prepare solutions with different concentrations: 0.1 mM, 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 5 mM, 6 mM; injecting a substrate into a capillary enzyme reactor by using an injection pump, collecting effluent liquid, and measuring the peak area of the product benzoyl ester-L-arginine by using a high-efficiency capillary electrophoresis apparatus;
the effective length of the eutectic solvent enzyme reactor is 5 cm, an empty capillary with the length of 50 cm and the inner diameter of 100 mu m is used for high-efficiency capillary electrophoresis analysis, the mobile phase is 20 mM Tris-HCl buffer solution with the pH value of 8.0, the separation voltage is 15 kV, and the detection wavelength is 214 nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811195644.5A CN109337811B (en) | 2018-10-15 | 2018-10-15 | Eutectic solvent monolithic column enzyme reactor and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811195644.5A CN109337811B (en) | 2018-10-15 | 2018-10-15 | Eutectic solvent monolithic column enzyme reactor and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109337811A CN109337811A (en) | 2019-02-15 |
| CN109337811B true CN109337811B (en) | 2021-10-12 |
Family
ID=65310050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811195644.5A Active CN109337811B (en) | 2018-10-15 | 2018-10-15 | Eutectic solvent monolithic column enzyme reactor and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109337811B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110124633B (en) * | 2019-04-29 | 2021-11-19 | 天津医科大学 | Monolithic column combining deep eutectic solvent monomer and hybrid monomer |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1383216A (en) * | 1972-05-03 | 1975-02-05 | Polymeric Enzymes Inc | Stabilization of enzymes |
| CN101687906A (en) * | 2007-05-16 | 2010-03-31 | 马里兰大学，大学城 | Reclaim the novel method of leaf protein |
| CN101776669A (en) * | 2010-02-07 | 2010-07-14 | 杭州师范大学 | Mixed matrix capillary electrochromatography monolithic column |
| CN104248863A (en) * | 2014-06-27 | 2014-12-31 | 南开大学 | Preparation method of aptamer affinity organic-silica hybrid capillary monolithic column |
| CN106832117A (en) * | 2015-12-03 | 2017-06-13 | 中国科学院大连化学物理研究所 | A kind of preparation method of organic whole material |
| CN106860399A (en) * | 2017-01-24 | 2017-06-20 | 天津医科大学 | The preparation of carbon nano tube-doped fenbufen molecularly imprinted polymer control slow-release material |
| CN108187367A (en) * | 2018-01-05 | 2018-06-22 | 福州大学 | Sulfydryl derivatization L-PROLINE type organic-inorganic hybridization monolithic column and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08267A (en) * | 1994-06-23 | 1996-01-09 | Nippon Shokubai Co Ltd | Immobilized biocatalyst |
-
2018
- 2018-10-15 CN CN201811195644.5A patent/CN109337811B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1383216A (en) * | 1972-05-03 | 1975-02-05 | Polymeric Enzymes Inc | Stabilization of enzymes |
| CN101687906A (en) * | 2007-05-16 | 2010-03-31 | 马里兰大学，大学城 | Reclaim the novel method of leaf protein |
| CN101776669A (en) * | 2010-02-07 | 2010-07-14 | 杭州师范大学 | Mixed matrix capillary electrochromatography monolithic column |
| CN104248863A (en) * | 2014-06-27 | 2014-12-31 | 南开大学 | Preparation method of aptamer affinity organic-silica hybrid capillary monolithic column |
| CN106832117A (en) * | 2015-12-03 | 2017-06-13 | 中国科学院大连化学物理研究所 | A kind of preparation method of organic whole material |
| CN106860399A (en) * | 2017-01-24 | 2017-06-20 | 天津医科大学 | The preparation of carbon nano tube-doped fenbufen molecularly imprinted polymer control slow-release material |
| CN108187367A (en) * | 2018-01-05 | 2018-06-22 | 福州大学 | Sulfydryl derivatization L-PROLINE type organic-inorganic hybridization monolithic column and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 《以低共熔溶剂为致孔剂制备分子印迹整体柱》;王涎桦等;《以低共熔溶剂为致孔剂制备分子印迹整体柱》;《第二十届全国色谱学术报告会及仪器展览会论文集》;20150419;第108-109页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109337811A (en) | 2019-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3325522B1 (en) | Polymeric particles | |
| KR102078892B1 (en) | Conjugated polymeric particle and method of making same | |
| CN102219800B (en) | Organic substituted boric acid ester, boron affinity functional material using organic substituted boric acid ester as functional monomer as well as preparation and application of organic substituted boric acid ester | |
| CN101565485B (en) | Method for preparing molecularly imprinted polymers of ethinylestradiol analogue | |
| CN103406109A (en) | Controllable and universal directional surface printing method and application of molecular printing polymer obtained via same | |
| CN108201795A (en) | A kind of preparation method of Selective Separation Enoxacin molecularly imprinted composite membrane material | |
| CN110115992B (en) | Aptamer functionalized polymer column for specific recognition of mycotoxin and preparation method thereof | |
| Xu et al. | Novel poly (vinyl alcohol)-based column coating for capillary electrophoresis of proteins | |
| CN109865507B (en) | Silica gel matrix surface modification method and application thereof | |
| CN109337811B (en) | Eutectic solvent monolithic column enzyme reactor and preparation method thereof | |
| CN106334343B (en) | A kind of preparation method and applications of agar sugar bonding silica-gel hydridization integral post | |
| CN108003287B (en) | Preparation method of protein affinity imprinted hydrogel polymer based on acrylamide metal chelating monomer | |
| CN104549183A (en) | Silica gel chromatography packing and preparation method thereof | |
| CN103833885A (en) | Micro-extraction monolithic column of ionic liquid polymer capillary tube and preparation method thereof | |
| Cheng et al. | Rapid proteolytic digestion and peptide separation using monolithic enzyme microreactor coupled with capillary electrophoresis | |
| Sibrian-Vazquez et al. | Improving the strategy and performance of molecularly imprinted polymers using cross-linking functional monomers | |
| CN104248863A (en) | Preparation method of aptamer affinity organic-silica hybrid capillary monolithic column | |
| CN105542076A (en) | Thiophilic porous material, and preparation method and application thereof | |
| CN110485165B (en) | Preparation and application of functionalized polyhedral oligomeric silsesquioxane modified polymer composite coating with specific enrichment function | |
| CN107474254B (en) | Preparation and application of organic-inorganic hydrophilic hybrid monolithic material | |
| CN101171075B (en) | Monolithic functionalisable materials | |
| CN109865315A (en) | A kind of organic-inorganic hybrid mesoporous material coating preparation method as chromatographic stationary phases | |
| CN106669637A (en) | Hydrophilic polymer stationary phase, and preparation method and application thereof | |
| CN102940980A (en) | Preparation method and application for hydrophilic organic polymer liquid phase monolithic chromatographic column | |
| CN105311858A (en) | Pentafluorobenzyl imidazolium salt ionic liquid hybrid monolithic column, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |