CN113527504A - Directional antibody immobilization method based on bioorthogonal reaction - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 35
- 150000001350 alkyl halides Chemical class 0.000 claims abstract description 32
- 239000000758 substrate Substances 0.000 claims abstract description 25
- 102000004190 Enzymes Human genes 0.000 claims abstract description 22
- 108090000790 Enzymes Proteins 0.000 claims abstract description 22
- 239000007790 solid phase Substances 0.000 claims abstract description 18
- 230000004927 fusion Effects 0.000 claims abstract description 15
- MLRVZFYXUZQSRU-UHFFFAOYSA-N 1-chlorohexane Chemical compound CCCCCCCl MLRVZFYXUZQSRU-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 230000000694 effects Effects 0.000 claims abstract description 6
- 239000004793 Polystyrene Substances 0.000 claims description 16
- 229920002223 polystyrene Polymers 0.000 claims description 16
- 238000005516 engineering process Methods 0.000 claims description 14
- 238000001179 sorption measurement Methods 0.000 claims description 10
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 238000007385 chemical modification Methods 0.000 claims description 9
- 230000003100 immobilizing effect Effects 0.000 claims description 7
- 238000003118 sandwich ELISA Methods 0.000 claims description 7
- 230000003213 activating effect Effects 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 230000035945 sensitivity Effects 0.000 claims description 5
- 238000008157 ELISA kit Methods 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000003259 recombinant expression Methods 0.000 claims description 4
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 239000007821 HATU Substances 0.000 claims description 3
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- FOCAUTSVDIKZOP-UHFFFAOYSA-N chloroacetic acid Chemical compound OC(=O)CCl FOCAUTSVDIKZOP-UHFFFAOYSA-N 0.000 claims description 3
- 229940106681 chloroacetic acid Drugs 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 238000001742 protein purification Methods 0.000 claims description 3
- 125000006850 spacer group Chemical group 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 4
- 238000001261 affinity purification Methods 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000000243 solution Substances 0.000 description 8
- 230000008569 process Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
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- 238000012827 research and development Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
- C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
- G01N2333/765—Serum albumin, e.g. HSA
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Abstract
The invention discloses a directed antibody immobilization method based on bio-orthogonal reaction, which comprises the following steps of introducing an enzyme with bio-orthogonal reaction activity as a label, and connecting the enzyme with an antibody to be immobilized in a fusion mode to obtain an antibody fused with the enzyme label. The high purity fusion enzyme labeled antibody is further reacted with alkyl halide dehalogenase substrate 6-chlorohexane to form covalent bonds to prepare a surface functionalized carrier surface. Incubating the antibody solution with the alkyl halide dehalogenase label with the surface of the solid phase carrier modified with the alkyl halide dehalogenase substrate, and realizing one-step covalent bond immobilization of the surface of the antibody functionalized solid phase carrier through the bioorthogonal reaction of the alkyl halide dehalogenase and the substrate 6-chlorohexane. The high specificity of bioorthogonal reaction is applied, the target antibody is fixed through a covalent bond, the fixed directionality and irreversibility are realized, the high stability is kept in different chemical and physical environments, and the method can be applied to the detection of a complex biological system and the sample affinity purification.
Description
Technical Field
The invention relates to the technical field of protein immobilization, in particular to a directional antibody immobilization method based on bioorthogonal reaction.
Background
The protein immobilization technology is the core of technologies such as biochips, microarrays, sensors, ELISA kits, separation and purification, and the like, antibodies are used as a special protein, and the antibody immobilization is also included in the technical category of protein immobilization. However, the most widely used method is the non-specific adsorption between the protein surface and the organic surface of the solid phase carrier, and the method has the advantages of low cost and simple operation, and only needs to simply incubate the protein solution and the solid phase carrier surface in a solution state. However, with the development of the next generation of biological detection technology, there are higher demands for precision, stability, reproducibility, etc., which depend on the efficiency of immobilization.
On the one hand, the immobilization of proteins or antibodies by non-specific adsorption has significant drawbacks: 1. on the molecular level, an immobilization interface, namely a contact surface between the surface of the solid phase carrier and the protein is not fixed and is irregular, so that a functional region of the protein or the antibody can be covered, and the efficiency of subsequent application is reduced; 2. the immobilization efficiency is poor, the nonspecific adsorption is easily influenced by the change of surrounding physical and chemical conditions, such as solution replacement, solution components, pH, temperature and other factors, and the nonspecific adsorption easily falls off from the surface of the solid phase carrier in the preparation or experiment process, so that the efficiency is reduced and the subsequent experiment result is unstable, and the requirements of latest scientific research and biotechnology research and development are difficult to meet.
On the other hand, although the introduction of affinity tags by recombinant protein expression techniques has been widely adopted, the commonly used histidine tag technique relies on electrostatic interactions between the tag and the metal ion resin, with some limitations: 1. although affinity and selectivity are improved compared to non-specific adsorption, many protein systems have significant surface charges, which cause interference and limit specificity to some extent; 2. the introduction of the metal ion system is easy to interfere with detection signals and influence the precision.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a directional antibody immobilization method based on bioorthogonal reaction.
In order to achieve the purpose, the invention adopts the technical scheme that: a directional antibody immobilization method based on bioorthogonal reaction comprises the following steps:
step S1: recombinant expression technology, enzyme with bio-orthogonal reaction activity is introduced as a label, and the label is connected with an antibody to be immobilized in a fusion mode to obtain an antibody of a fusion enzyme label.
Step S2: and (3) activating the surface of the polystyrene, and further reacting the antibody of the high-purity fusion enzyme label with an alkyl halide dehalogenase substrate 6-chlorohexane to form a covalent bond to prepare the surface functionalized carrier surface.
Step S3: incubating the antibody solution with the alkyl halide dehalogenase label with the surface of the solid phase carrier modified with the alkyl halide dehalogenase substrate, and realizing one-step covalent bond immobilization of the surface of the antibody functionalized solid phase carrier through the bioorthogonal reaction of the alkyl halide dehalogenase and the substrate 6-chlorohexane.
Step S4: and (3) characterizing and verifying the density and distribution of surface modification through a scanning electron microscope, surface element analysis and a fluorescence microscope.
In a preferred embodiment of the present invention, in the step S1, the high-purity antibody labeled with the fusion enzyme is obtained by a protein purification technique.
In a preferred embodiment of the present invention, in step S1, alkyl halide dehalogenase is selected as an immobilized tag to be fused to the N-terminal of the antibody.
In a preferred embodiment of the present invention, in step S1, the antibody and the alkyl halide dehalogenase are linked by GS spacer arms of different lengths, and the whole expression and purification are performed.
In a preferred embodiment of the present invention, after the step S4, a product test is performed, and a sandwich ELISA experiment is designed by selecting a monoclonal antibody against bovine serum albumin BSA as a target.
In a preferred embodiment of the present invention, the detection sensitivity, specificity, accuracy and stability of the sandwich ELISA kit based on the coated antibody directional immobilization technology for BSA complex samples in the range of 0-100 ng were tested and compared with those of conventional ELISA kits prepared by non-specific adsorption methods.
In a preferred embodiment of the present invention, during the step S2 of activating the polystyrene surface, the polystyrene surface is subjected to an ultraviolet irradiation treatment in an ozone environment.
In a preferred embodiment of the present invention, in step S2, the polystyrene surface is activated to form a hydroxyl surface, and the substrate for the alkylhalide dehalogenase is attached by two-step chemical modification.
In a preferred embodiment of the present invention, the first step of chemical modification is carried out by adding aminopropyltriethoxysilane and toluene at room temperature for 1-3 hours.
In a preferred embodiment of the present invention, the second step of chemical modification is carried out by adding chloroacetic acid, DEA, DMF and HATU and reacting at room temperature for 1-3 hours.
The invention solves the defects in the background technology, and has the following beneficial effects:
(1) the invention is based on the immobilization technology of bioorthogonal reaction, applies the high specificity of bioorthogonal reaction, fixes the target antibody through a covalent bond, realizes the directionality and the irreversibility of immobilization, keeps high stability in different chemical and physical environments, can be applied to the detection and the sample affinity purification of a complex biological system, and has better efficiency of subsequent application.
(2) According to the invention, through mutual recognition of high-specificity enzyme and substrate and generation of chemical reaction process covalent bond, the antibody is connected to the surface of the solid phase carrier through the covalent bond, compared with a system of non-specific adsorption and mutual recognition through electrostatic force, the covalent bond has extremely high stability and specificity, is not influenced by a subsequent preparation process or an experimental process, is a powerful guarantee on sensitivity, accuracy and reproducibility, can be reused, prolongs the service life of an extended product, and reduces the use cost on the whole.
(3) According to the invention, a substrate connected with Halo enzyme is chemically modified in two steps, an antibody carrying an alkyl halide dehalogenase label is expressed in a recombination manner, and reacts with the surface of an active solid phase carrier connected with the alkyl halide dehalogenase substrate to form a covalent bond, so that one-step covalent immobilization of the antibody is realized, and the antibody can be applied to ELISA and biosensor development.
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 described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of a process for the surface modification of alkyl halide dehalogenase substrates to polystyrene in accordance with a preferred embodiment of the present invention;
FIG. 2 is a schematic diagram of the procedure for the immobilization of the alkylhalogenodehalogenase tag in a preferred embodiment of the present invention.
Detailed Description
In order that the above objects, features and advantages of the present invention can be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
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. While various other embodiments of the present invention will become apparent to those skilled in the art from the following description, which is given without intending to be inventive, the invention is not limited to the specific embodiments disclosed below, since many more specific details are set forth in the following description in order to provide a thorough understanding of the invention, but the invention can be practiced in other ways than those specifically described herein.
As shown in fig. 1 and fig. 2, a method for immobilizing a directed antibody based on bio-orthogonal reaction includes the following steps:
step S1: recombinant expression technology, enzyme with bio-orthogonal reaction activity is introduced as a label, and the label is connected with an antibody to be immobilized in a fusion mode to obtain an antibody of a fusion enzyme label.
Step S2: and (3) activating the surface of the polystyrene, and further reacting the antibody of the high-purity fusion enzyme label with an alkyl halide dehalogenase substrate 6-chlorohexane to form a covalent bond to prepare the surface functionalized carrier surface.
Step S3: incubating the antibody solution with the alkyl halide dehalogenase label with the surface of the solid phase carrier modified with the alkyl halide dehalogenase substrate, and realizing one-step covalent bond immobilization of the surface of the antibody functionalized solid phase carrier through the bioorthogonal reaction of the alkyl halide dehalogenase and the substrate 6-chlorohexane.
Step S4: and (3) characterizing and verifying the density and distribution of surface modification through a scanning electron microscope, surface element analysis and a fluorescence microscope.
In a preferred embodiment of the invention, the immobilization technology based on the bioorthogonal reaction is applied, the high specificity of the bioorthogonal reaction is applied, the target antibody is immobilized through a covalent bond, the immobilization directionality and the irreversibility are realized, the high stability is kept in different chemical and physical environments, the method can be applied to the detection and the sample affinity purification of a complex biological system, and the efficiency of the subsequent application is better.
In a preferred embodiment of the invention, the antibody is connected to the surface of the solid phase carrier by a covalent bond through mutual recognition of a high-specificity enzyme and a substrate and a chemical reaction process covalent bond, compared with a system of non-specific adsorption and mutual recognition through electrostatic force, the covalent bond has extremely high stability and specificity, is not influenced by a subsequent preparation process or an experimental process, is a powerful guarantee on sensitivity, accuracy and reproducibility, can realize repeated use, prolongs the service life of an extended product, and reduces the use cost on the whole.
In a preferred embodiment of the present invention, during the activation of the polystyrene surface in step S2, the polystyrene surface is subjected to ultraviolet irradiation treatment in an ozone environment to perform a certain sterilization and disinfection effect, so as to satisfy the reaction conditions for activating the polystyrene surface and effectively improve the activation effect of the polystyrene surface.
In a preferred embodiment of the present invention, a sandwich ELISA experiment is designed by using a monoclonal antibody against bovine serum albumin BSA as an object. The detection sensitivity, specificity, accuracy and stability of the sandwich ELISA kit based on the coated antibody directional immobilization technology to a BSA complex sample in the range of 0-100 nanogram are tested, and compared with the conventional ELISA kit prepared by a non-specific adsorption method, the sandwich ELISA kit can better improve the product so as to maximize the product efficiency.
In a preferred embodiment of the present invention, in step S2, the polystyrene surface is activated to form a hydroxyl surface, and the substrate for the alkylhalodehalogenase is attached by two-step chemical modification. The first step of chemical modification reaction is carried out by adding aminopropyltriethoxysilane and toluene and reacting at room temperature for 1-3 hours, preferably 2 hours. The second step of chemical modification reaction conditions are that chloroacetic acid, DEA, DMF and HATU are added to react for 1 to 3 hours, preferably for 2 hours at room temperature; the method is characterized in that a substrate connected with Halo enzyme is chemically modified in two steps, an antibody carrying an alkyl halide dehalogenase label is expressed in a recombination mode, and reacts with the surface of an active solid phase carrier connected with the alkyl halide dehalogenase substrate to form a covalent bond, so that one-step covalent immobilization of the antibody is realized, and the antibody can be applied to ELISA and biosensor development.
When the fusion protein is used, the enzyme with bio-orthogonal reactivity is introduced as a label by a recombinant expression technology, the enzyme is connected with an antibody needing to be immobilized in a fusion mode, the antibody of the high-purity fusion enzyme label is obtained by a protein purification technology, the alkyl halide dehalogenase is specifically selected as an immobilized label and fused to the N tail end of the antibody, and the antibody and the alkyl halide dehalogenase are connected through GS (glutathione) spacer arms with different lengths to carry out integral expression and purification. And (3) activating the surface of the polystyrene, and further reacting the antibody of the high-purity fusion enzyme label with an alkyl halide dehalogenase substrate 6-chlorohexane to form a covalent bond to prepare the surface functionalized carrier surface. Incubating the antibody solution with the alkyl halide dehalogenase label with the surface of the solid phase carrier modified with the alkyl halide dehalogenase substrate, and realizing one-step covalent bond immobilization of the surface of the antibody functionalized solid phase carrier through the bioorthogonal reaction of the alkyl halide dehalogenase and the substrate 6-chlorohexane. And (3) characterizing and verifying the density and distribution of surface modification through a scanning electron microscope, surface element analysis and a fluorescence microscope.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. A directional antibody immobilization method based on bioorthogonal reaction is characterized by comprising the following steps:
step S1: recombinant expression technology, introducing enzyme with bio-orthogonal reaction activity as a label, and connecting the label with an antibody to be fixed in a fusion mode to obtain an antibody fused with the enzyme label;
step S2: activating the surface of polystyrene, and further reacting the antibody of the high-purity fusion enzyme label with an alkyl halide dehalogenase substrate 6-chlorohexane to form a covalent bond to prepare a surface functionalized carrier surface;
step S3: incubating an antibody solution with an alkyl halide dehalogenase label with the surface of a solid phase carrier modified with an alkyl halide dehalogenase substrate, and realizing one-step covalent bond immobilization of the surface of the antibody functionalized solid phase carrier through a bioorthogonal reaction of alkyl halide dehalogenase and the substrate 6-chlorohexane;
step S4: and (3) characterizing and verifying the density and distribution of surface modification through a scanning electron microscope, surface element analysis and a fluorescence microscope.
2. The method for immobilizing a directional antibody based on bioorthogonal reaction of claim 1, wherein: in the step S1, a high-purity fusion enzyme-tagged antibody is obtained by a protein purification technique.
3. The method for immobilizing a directional antibody based on bioorthogonal reaction of claim 1, wherein: in step S1, an alkyldehalogenase is selected as the immobilized tag to be fused to the N-terminus of the antibody.
4. The method for immobilizing a directional antibody based on bioorthogonal reaction of claim 1, wherein: in step S1, the antibody and the alkyl halide dehalogenase are ligated by GS spacer arms of different lengths, and the whole is expressed and purified.
5. The method for immobilizing a directional antibody based on bioorthogonal reaction of claim 1, wherein: and (3) performing product test after the step S4, and designing a sandwich ELISA experiment by using a monoclonal antibody against bovine serum albumin BSA as an object.
6. The method of claim 5, wherein the method comprises the following steps: the detection sensitivity, specificity, accuracy and stability of the sandwich ELISA kit based on the coated antibody directional immobilization technology to BSA complex samples in the range of 0-100 nanogram are tested and compared with the conventional ELISA kit prepared by a non-specific adsorption method.
7. The method for immobilizing a directional antibody based on bioorthogonal reaction of claim 1, wherein: upon activation of the polystyrene surface in the step S2, the polystyrene surface is subjected to ultraviolet irradiation treatment in an ozone atmosphere.
8. The method for immobilizing a directional antibody based on bioorthogonal reaction of claim 1, wherein: in step S2, the polystyrene surface is activated to form a hydroxyl surface, and the substrate for the alkylhalide dehalogenase is attached by two-step chemical modification.
9. The method of claim 8, wherein the method comprises: the first step of chemical modification reaction is to add aminopropyl triethoxy silane and toluene to react for 1-3 hours at room temperature.
10. The method of claim 8, wherein the method comprises: the second step of chemical modification reaction is to add chloroacetic acid, DEA, DMF and HATU to react for 1-3 hours at room temperature.
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|---|---|---|---|---|
| CN102040653A (en) * | 2010-09-07 | 2011-05-04 | 湘潭大学 | Tag peptide capable of realizing affinity binding with polystyrene and method for preparing enzyme-linked immuno solid phase antigen with same |
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| CN111978404A (en) * | 2020-08-11 | 2020-11-24 | 西北大学 | Protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction |
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- 2021-08-10 CN CN202110912619.XA patent/CN113527504A/en active Pending
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|---|---|---|---|---|
| CN102040653A (en) * | 2010-09-07 | 2011-05-04 | 湘潭大学 | Tag peptide capable of realizing affinity binding with polystyrene and method for preparing enzyme-linked immuno solid phase antigen with same |
| CN102998444A (en) * | 2012-07-15 | 2013-03-27 | 潍坊医学院 | Stereotactic fixing method of IgG antibody on surface of polystyrene carrier |
| WO2014140300A1 (en) * | 2013-03-15 | 2014-09-18 | Innate Pharma | Solid phase tgase-mediated conjugation of antibodies |
| WO2017027337A1 (en) * | 2015-08-13 | 2017-02-16 | The Regents Of The University Of California | Bioorthogonal reaction of an amine n-oxide and a boron agent |
| CN111978404A (en) * | 2020-08-11 | 2020-11-24 | 西北大学 | Protein one-step directional immobilization method based on Halo-tag specific dehalogenation reaction |
Non-Patent Citations (1)
| Title |
|---|
| KAIZHU ZENG ET AL.: "One-step methodology for the direct covalent capture of GPCRs from complex matrices onto solid surfaces based on the bioorthogonal reaction between haloalkane dehalogenase and chloroalkanes", 《CHEMICAL SCIENCE》 * |
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