CN112048045B - Preparation method of sensitive western blotting material based on block macromolecular chain monomer - Google Patents
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- C08J2489/00—Characterised by the use of proteins; Derivatives thereof
Abstract
The invention discloses a preparation method of a sensitive western blotting material based on a block macromolecular chain monomer, which comprises the following steps: step 1, dissolving O-ethyl-S- (1-phenethyl) dithiocarbonate and a sensitive monomer in an organic solvent, and reacting under an anaerobic condition to obtain a block macromolecular chain precursor 1; step 2, respectively adding a block macromolecular chain precursor 1, hydroxyethyl acrylate, a zwitterionic monomer and an initiator into an organic solvent, and reacting under an anaerobic condition to obtain a block copolymerization macromolecular chain precursor 2; step 3, reacting the obtained block copolymerization macromolecular chain precursor 2 with acryloyl chloride to obtain a macromolecular functional monomer; and 4, placing the protein and the macromolecular functional monomer into a phosphoric acid buffer solution for assembly, and adding ammonium persulfate and tetramethylethylenediamine to obtain the western blotting hydrogel. The macromolecular chain monomer can obtain an imprinted hole structure with high specificity interaction in the molecularly imprinted material.
Description
Technical Field
The invention belongs to the technical field of high polymer materials, and relates to a preparation method of a sensitive protein imprinting material based on a block macromolecular chain monomer.
Background
Proteomics is a study of the characteristics of proteins on different scales, including protein expression, protein translation, and protein-protein interaction, and has been receiving extensive attention in recent years. By comparing proteomes of healthy individuals and pathological individuals, several protein biomarkers associated with the pathology were found. These proteins can be used as new molecular targets for drug design or molecular markers for early diagnosis of diseases. In order to isolate or analyze a target protein, an immunoassay based on an antigen-antibody specific recognition effect is considered to be the most effective method. However, the screening and production of antibodies are complex, the cost is high, the stability is poor, and the storage is not easy, so that the high requirements of the modern biological industry on the yield and the purity of protein preparations are difficult to meet. Therefore, there is a need to develop an efficient strategy for selectively isolating proteins from complex samples
The advent of Molecularly Imprinted polymer Materials (MIPs) prepared by covalent or non-covalent interactions of high molecular weight Polymers with template molecules has made this possible. The intelligent recognition effect of the molecularly imprinted polymer material on the template molecules is mainly caused by the existence of imprinted holes which have the function of mutually matching the shape, size and functional groups of the template molecules in the polymer, and the technology is put forward by scholars at home and abroad. Compared with the imprinting technology using small molecules as templates, the western imprinting still faces many challenges due to the characteristics of large volume, structural variability, abundant surface functional groups and the like of proteins. Related researches show that the transfer efficiency of protein can be effectively improved by using a stimulus-responsive imprinted polymer as MIPs. Xie et al prepared a western-imprinted polymer with dual photon and magnetic response capability by a surface imprinting method for specific recognition of BHb. Experimental results show that the MIPs have good combination behavior on BHb, fast combination kinetics, large adsorption capacity and good selectivity. MIPs demonstrated good photocontrol of the templating protein BHb under alternating 365 and 440nm illumination, at 365nm31.4% of BHb is released from MIPs, and 67.2% of BHb is incorporated into MIPs at 440 nm. Thus, MIPs have been successfully applied to the extraction of template proteins from actual biological samples (Xie X, Hu Q, Ke R, et al. factor prediction of photonic and magnetic passive protein for specific biological recognition of bone hemoglobin [ J]Chemical Engineering Journal,2019,371: 130-. Kitayama et al successfully synthesized novel gas-stimuli-responsive nuclear shell microparticles having molecular recognition ability for the first time. Control of affinity for target proteins by introduction of gases with selective binding properties, and N2Compared with the treatment, the MIP particles are in CO2Exhibit higher affinity for the target protein in treated aqueous media while maintaining better selectivity for the template protein (Kitayama, Yukiya, Isomura, et. gas-viscous-reactive molecular imprinted polymer particles with switched affinity for target protein [ J]Chemical Communications, 2018.). Qin et al introduce thermal response characteristics into surface imprinting sites, thereby regulating the 'adsorption and release' behavior of a Protein imprinting polymer through temperature change, so as to prepare a novel Thermosensitive Protein imprinting Hydrogel, selectively identifying lysozyme through self-assembly with chelating monomers and copper ions, and the result shows that the imprinting Hydrogel has the lysozyme identification capacity and the imprinting factor of up to 7.87(Qin L, He X W, Zhang W, et al]Analytical Chemistry,2009,81(17): 7206-. In addition, Krysico believes that the structural failure phenomenon of proteins during imprinting is mainly caused by the failure of functional monomers and cross-linking agents (Krysio D R, flight M Q, Peppas N A. protein formatting for macromolecular imprinted polymers [ J]Macromolecular Bioscience,2012,12(8):1137-1144), traditional functional monomers and cross-linking agents impart their flexibility to penetrate more easily into the interior of proteins relative to smaller molecular volumes of proteins, thus disrupting the hydrogen bonds that maintain structural and conformational stability within the protein, resulting in changes in the structure of the protein. On the other hand, the protein has complex structure and rich surfaceThe amino acid residues of different properties inevitably react with the surface of the material other than the imprinted pores, resulting in nonspecific adsorption and a decrease in the recognition ability of the imprinted material. The zwitterion material is a novel protein inert acting material, and a water shell layer is formed on the surface of the material through the hydration of zwitterion functional groups and water molecules, so that the interaction between protein and the material can be effectively blocked.
Disclosure of Invention
The invention aims to provide a preparation method of a sensitive western blotting material based on a block macromolecular chain monomer.
The technical scheme adopted by the invention is that the preparation method of the sensitive western blotting material based on the block macromolecular chain monomer specifically comprises the following steps:
step 1, dissolving O-ethyl-S- (1-phenethyl) dithiocarbonate and a sensitive monomer in an organic solvent, adding an initiator, and reacting under an anaerobic condition to obtain a block macromolecular chain precursor 1;
step 2, respectively adding a block macromolecular chain precursor 1, hydroxyethyl acrylate, a zwitterionic monomer and an initiator into an organic solvent, and reacting under an anaerobic condition to obtain a block copolymerization macromolecular chain precursor 2;
step 3, reacting the block copolymerization macromolecular chain precursor 2 obtained in the step 2 with acryloyl chloride to obtain a macromolecular functional monomer;
and 4, placing the protein and the macromolecular functional monomer obtained in the step 3 into a phosphoric acid buffer solution for assembly, sequentially adding ammonium persulfate and tetramethylethylenediamine, and reacting under an anaerobic condition to obtain the western blotting hydrogel.
The present invention is also characterized in that,
in the step 1, the sensitive monomer is one of 4- [ (4-methacryloyloxy) phenylazo ] benzenesulfonic acid, N, N-dimethylaminoethyl methacrylate and isopropylacrylamide.
In the step 1, the molar ratio of the O-ethyl-S- (1-phenethyl) dithiocarbonate to the sensitive monomer is 1: 1-20.
In step 2, the zwitterionic monomer is one of N, N-dimethyl (methacryloyloxyethyl) ammonio propanesulfonate inner salt, methacryloyloxyethyl phosphorylcholine and sulfobetaine methacrylate.
In the step 2, the molar ratio of the block macromolecular chain precursor 1 to the zwitterionic monomer is 1: 10-100.
In the step 2, the molar ratio of the block macromolecular chain precursor 1 to the hydroxyethyl acrylate is 1: 10-100.
The mol ratio of the block copolymerization macromolecular chain precursor 2 to the acryloyl chloride in the step 3 is 1: 10-100.
The mass ratio of the protein to the macromolecular functional monomer in the step 4 is 1: 1-10.
The organic solvent in the step 1 is N, N-dimethyl sulfoxide; the initiator is azobisisobutyronitrile.
The invention has the beneficial effects that based on the structural characteristics of protein, the invention designs and prepares the block copolymerization macromolecular chain capable of maintaining the structural stability of the protein as a functional monomer of the protein imprinting, the functional area of the macromolecular chain monomer is composed of monomers which can generate good interaction with the protein and are sensitive to external conditions, the cross-linking area contains protein pollution resistant amphoteric polymer which can effectively reduce the nonspecific adsorption of the material, and the monomer provides an effective method for the high-identification protein imprinting material.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
The preparation method of the sensitive western blotting material based on the block macromolecular chain monomer realizes the construction of the photosensitive high-identification western blotting material simultaneously comprising the functional region and the crosslinking region through the design of the block macromolecular chain monomer. The method specifically comprises the following steps:
preparation of block macromolecular chain monomer
1) Dissolving a certain amount of O-ethyl-S- (1-phenylethyl) dithiocarbonate as a Chain Transfer Agent (CTA) and a certain amount of sensitive monomer in 10-30 mL of N, N-dimethyl sulfoxide (DMSO) solution (ensuring that the concentration of the total monomer is 2-20% mg/mL), and then adding a certain amount of azobisisobutyronitrile initiator (AIBN initiator accounts for 0.5-2% wt of the total monomer) into the mixed solution. Wherein, the molar ratio of CTA and sensitive monomer is 1: 10-250.
2) Introducing nitrogen into the mixed solution to remove oxygen for 30min, and reacting at 60-80 ℃ for 1-18 h. The reacted solution was precipitated in 50-100mL of iced ether and washed several times with 200mL of ether. And drying the precipitated product at room temperature of 25 ℃ for 12h under vacuum condition to obtain a precursor 1 of the block macromolecular chain.
3) 200mg of the block macromolecular chain precursor 1 is dissolved in a certain amount of DMSO (ensuring that the concentration of the total monomer is 2-20% mg/mL), and then a certain amount of zwitterionic monomer, hydroxyethyl acrylate (HEA) and azobisisobutyronitrile initiator (the weight of AIBN initiator accounts for 0.5-2% wt of the total monomer) are added into the solution. Wherein the molar ratio of the precursor 1 to the zwitterionic monomer and HEA is 1: 10-100.
4) Introducing nitrogen into the functional macromolecular chain mixed solution, deoxidizing for 30min, and reacting for 2-24h at 60-80 ℃. The reacted solution was precipitated in 100mL of ethyl ether and washed several times with 200mL of ethyl ether. And drying the precipitated product at room temperature of 25 ℃ for 12h under a vacuum condition to obtain a block macromolecular chain precursor 2.
5) In DMSO, reacting the obtained macromolecular chain precursor 2 with acryloyl chloride and azobisisobutyronitrile initiator at 30-80 ℃ for 12-50h to obtain the final macromolecular chain. The precipitated crude product was dissolved in 10mL of deionized water, and the aqueous solution was placed in a dialysis bag with a molecular weight cut-off of 1000 for dialysis for 2 days. And (4) freeze-drying the dialyzed aqueous solution to obtain the final block macromolecular chain monomer.
Preparation of protein imprinted hydrogel based on block macromolecular chain monomer
1) 100mg of protein and 100-500 mg of block copolymerization macromolecular chain monomer are put into 1-10 mL (ensuring the solid content of gel is 5-30 wt%) of phosphate buffer solution (pH 7, 10mM) for assembly for 2 h.
2) Then, an amount of ammonium persulfate was dissolved in the above solution, and oxygen was removed by introducing nitrogen for 30 min. A certain amount of tetramethylethylenediamine was quickly added to the solution. The solution reacts for 2-24 hours at 20-30 ℃.
Wherein the ammonium persulfate and the tetramethyl ethylene diamine account for 0.5-2% wt of the total monomer mass, and the ratio of the ammonium persulfate to the tetramethyl ethylene diamine is 1:1 (milligram: microliter)
3) Washing the generated imprinted hydrogel in a NaCl solution with the concentration range of 0.1-0.5M at 20-30 ℃, measuring the characteristic peak of the eluent at 280nm by using an ultraviolet spectrophotometer, and continuing the washing process until the characteristic peak at 280nm cannot be observed in the ultraviolet spectrum of the eluent. The NaCl on the hydrogel surface was then eluted with 500mL of deionized water. And drying the obtained imprinted hydrogel in a vacuum oven at 30 ℃ for 24-48 h. The preparation of non-imprinted hydrogel (NIH) was identical to the preparation of the imprinted hydrogel (MIH) described above, except that no template protein was added.
Various embodiments are enumerated
Example 1
The synthesis process of the photosensitive western blotting material obtained in this example is as follows:
preparation of block macromolecular chain monomer
1) 226mg of O-ethyl-S- (1-phenylethyl) dithiocarbonate as a chain transfer agent (CTA, Mn: 226g/mol) and 3460mg of 4- [ (4-methacryloyloxy) phenylazo ] benzenesulfonic acid were dissolved in 10mL of a solution of N, N-Dimethylsulfoxide (DMSO), followed by addition of 2.36mg of azobisisobutyronitrile initiator to the mixed solution.
2) And introducing nitrogen into the mixed solution to remove oxygen for 30min, and reacting at 60 ℃ for 1 h. The reacted solution was precipitated in 50mL of ethyl ether and washed several times with 200mL of ethyl ether. Drying the precipitated product for 12 hours under the vacuum condition at normal temperature to obtain a block macromolecular chain precursor 1, wherein the synthesis process of the block macromolecular chain precursor 1 is as follows:
3) 500mg of block macromolecular chain precursor 1 was dissolved in 10mL of DMSO, and then 300mg of N, N-dimethyl (methacryloyloxyethyl) ammonio propanesulfonate inner salt (DMAPS), 300mg of hydroxyethyl acrylate (HEA) and 6mg of azobisisobutyronitrile initiator were added to the solution.
4) The mixed solution obtained is deaerated for 30min by introducing nitrogen and then reacted for 2h at 60 ℃. The reacted solution was precipitated in 100mL of iced ether and washed with 200mL of ether several times. Drying the precipitated product for 12 hours under the vacuum condition at normal temperature to obtain a block macromolecular chain precursor 2, wherein the synthesis process of the block macromolecular chain precursor 2 is as follows:
5) in DMSO, the obtained 500mg of macromolecular chain precursor 2, 100mg of acryloyl chloride and azobisisobutyronitrile initiator react for 12 hours at 30 ℃ to obtain the final macromolecular chain. The precipitated crude product was dissolved in 10mL of deionized water, and the aqueous solution was placed in a dialysis bag with a molecular weight cut-off of 1000 for dialysis for 2 days. And (3) freeze-drying the dialyzed aqueous solution to obtain a final block macromolecular chain monomer, wherein the synthesis process of the block macromolecular chain monomer is as follows:
preparation of protein imprinted hydrogel based on block macromolecular chain monomer
1) 100mg of protein was assembled with 100mg of functional macromolecular chains in 4mL of phosphate buffered saline (pH 7, 10mM) for 2 h.
2) Subsequently, 8mg of ammonium persulfate was dissolved in the above solution, and oxygen was removed by introducing nitrogen gas for 30 min. To the solution was added 8. mu.L of tetramethylethylenediamine quickly. The solution was reacted at 20 ℃ for 2 h.
3) The generated imprinted hydrogel is washed in NaCl solution with the concentration range of 0.1M at the temperature of 20 ℃, the characteristic peak of the eluent at 280nm is measured by an ultraviolet spectrophotometer, and the washing process is continued until the characteristic peak at 280nm cannot be observed in the ultraviolet spectrum of the eluent. Then, 1L of deionized water was used to elute NaCl from the surface of the hydrogel. The resulting blotting hydrogel was dried in a vacuum oven at 30 ℃ for 24 h.
Example 2
The synthesis process of the gas-sensitive western blotting material obtained in this example is as follows:
preparation of block macromolecular chain monomer
1) 214mg of CTA and 3140mg of N, N-dimethylaminoethyl methacrylate were dissolved in 20mL of N, N-dimethyl sulfoxide (DMSO) solution, followed by adding 2.64mg of azobisisobutyronitrile initiator to the mixed solution.
2) And introducing nitrogen into the mixed solution to remove oxygen for 30min, and reacting at 70 ℃ for 3 h. The reacted solution was precipitated in 80mL of iced ether and washed several times with 200mL of ether. Drying the precipitated product for 12h under the vacuum condition at normal temperature to obtain a block macromolecular chain precursor 1, wherein the synthesis process of the block macromolecular chain precursor 1 is as follows:
3) 500mg of the block macromolecular chain precursor 1 was dissolved in 10mL of DMSO, and then 150mg of Methacryloyloxyethyl Phosphorylcholine (MPC), 150mg of hydroxyethyl acrylate (HEA), and 5mg of azobisisobutyronitrile initiator were added to the solution.
4) The mixed solution obtained is deaerated for 30min by introducing nitrogen and then reacted for 3h at 70 ℃. The reacted solution was precipitated in 100mL of ethyl ether and washed several times with 200mL of ethyl ether. Drying the precipitated product for 12h under the vacuum condition at normal temperature to obtain a block macromolecular chain precursor 2, wherein the synthesis process of the block macromolecular chain precursor 2 is as follows:
5) in DMSO, the obtained 500mg of macromolecular chain precursor 2, 200mg of acryloyl chloride and 9mg of azobisisobutyronitrile initiator are reacted for 36 hours at 60 ℃ to obtain the final macromolecular chain. The precipitated crude product was dissolved in 10mL of deionized water, and the aqueous solution was placed in a dialysis bag with a molecular weight cut-off of 1000 for dialysis for 2 days. And (3) freeze-drying the dialyzed aqueous solution to obtain a final block macromolecular chain monomer, wherein the synthesis process of the block macromolecular chain monomer is as follows:
preparation of protein imprinted hydrogel based on block macromolecular chain monomer
1) 100mg of protein was assembled with 300mg of functional macromolecular chains in 5mL of phosphate buffered saline (pH 7, 10mM) for 2 h.
2) Then 9mg of ammonium persulfate was dissolved into the above solution, and oxygen was removed by nitrogen introduction for 30 min. 9 μ L of tetramethylethylenediamine was added quickly to the solution. The solution was reacted at 25 ℃ for 6 h.
3) The generated imprinted hydrogel is washed in NaCl solution with the concentration range of 0.3M at 25 ℃, the characteristic peak of the eluent at 280nm is measured by an ultraviolet spectrophotometer, and the washing process is continued until the characteristic peak at 280nm cannot be observed in the ultraviolet spectrum of the eluent. Subsequently, 500mL of deionized water was used to elute NaCl from the hydrogel surface. The resulting blotting hydrogel was dried in a vacuum oven at 30 ℃ for 36 h.
Example 3
The synthesis process of the temperature-sensitive western blotting material obtained in this example is as follows:
preparation of block macromolecular chain monomer
1) 214mg of CTA and 28000mg of isopropylacrylamide were dissolved in 30mL of a N, N-dimethyl sulfoxide (DMSO) solution, and 141.07mg of azobisisobutyronitrile initiator was added to the mixed solution.
2) And introducing nitrogen into the mixed solution to remove oxygen for 30min, and reacting at 80 ℃ for 18 h. The reacted solution was precipitated in 100mL of ethyl ether and washed several times with 200mL of ethyl ether. And drying the precipitated product for 12h under the vacuum condition at normal temperature to obtain the block macromolecular chain precursor 1.
3) 500mg of the block macromolecular chain precursor 1 was dissolved in 10mL of DMSO, and then 150mg of sulfobetaine methacrylate (SBMA), 200mg of hydroxyethyl acrylate (HEA), and 5mg of azobisisobutyronitrile initiator were added to the solution.
4) The mixed solution obtained is introduced with nitrogen to remove oxygen for 30min, and then reacted for 24h at 80 ℃. The reacted solution was precipitated in 100mL of ethyl ether and washed several times with 200mL of ethyl ether. And drying the precipitated product for 12 hours under the vacuum condition at normal temperature to obtain a segmented macromolecular chain precursor 2.
5) In DMSO, the obtained 500mg of macromolecular chain precursor 2 reacts with 300mg of acryloyl chloride and 8mg of azobisisobutyronitrile initiator at 80 ℃ for 50h to obtain the final macromolecular chain. The precipitated crude product was dissolved in 10mL of deionized water, and the aqueous solution was placed in a dialysis bag with a molecular weight cut-off of 1000 for dialysis for 2 days. And (4) freeze-drying the dialyzed aqueous solution to obtain the final block macromolecular chain monomer.
Preparation of protein imprinted hydrogel based on block macromolecular chain monomer
1) 100mg of protein was assembled with 500mg of functional macromolecular chains in 10mL of phosphate buffered saline (pH 7, 10mM) for 2 h.
2) Subsequently, 8mg of ammonium persulfate was dissolved in the above solution, and oxygen was removed by introducing nitrogen for 30 min. 10 μ L of tetramethylethylenediamine was added quickly to the solution. The solution was reacted at 30 ℃ for 24 h.
3) The generated imprinted hydrogel is washed in NaCl solution with the concentration range of 0.5M at 30 ℃, the characteristic peak of the eluent at 280nm is measured by an ultraviolet spectrophotometer, and the washing process is continued until the characteristic peak at 280nm cannot be observed in the ultraviolet spectrum of the eluent. Subsequently, 500mL of deionized water was used to elute NaCl from the hydrogel surface. The resulting blotting hydrogel was dried in a vacuum oven at 30 ℃ for 48 h.
Claims (9)
1. The preparation method of the sensitive western blotting material based on the block macromolecular chain monomer is characterized in that: the method specifically comprises the following steps:
step 1, dissolving O-ethyl-S- (1-phenethyl) dithiocarbonate and a sensitive monomer in an organic solvent, adding an initiator, and reacting under an anaerobic condition to obtain a block macromolecular chain precursor 1;
step 2, respectively adding a block macromolecular chain precursor 1, hydroxyethyl acrylate, a zwitterionic monomer and an initiator into an organic solvent, and reacting under an anaerobic condition to obtain a block copolymerization macromolecular chain precursor 2;
step 3, reacting the block copolymerization macromolecular chain precursor 2 obtained in the step 2 with acryloyl chloride to obtain a macromolecular functional monomer;
and 4, placing the protein and the macromolecular functional monomer obtained in the step 3 into a phosphoric acid buffer solution for assembly, sequentially adding ammonium persulfate and tetramethylethylenediamine, and reacting under an anaerobic condition to obtain the western blotting hydrogel.
2. The method for preparing the sensitive western blotting material based on the block macromolecular chain monomer according to claim 1, characterized in that: in the step 1, the sensitive monomer is one of 4- [ (4-methacryloxy) phenylazo ] benzenesulfonic acid, N, N-dimethylaminoethyl methacrylate and isopropyl acrylamide.
3. The method for preparing a sensitive western blotting material based on a block macromonomer according to claim 2, characterized in that: in the step 1, the molar ratio of the O-ethyl-S- (1-phenethyl) dithiocarbonate to the sensitive monomer is 1: 1-20.
4. The method for preparing the sensitive western blotting material based on the block macromolecular chain monomer according to claim 1, characterized in that: in the step 2, the zwitterion monomer is one of N, N-dimethyl (methacryloxyethyl) ammonio propanesulfonate inner salt, methacryloxyethyl phosphorylcholine and sulfobetaine methacrylate.
5. The method for preparing the sensitive western blotting material based on the block macromolecular chain monomer according to claim 4, characterized in that: in the step 2, the molar ratio of the segmented macromolecular chain precursor 1 to the zwitterionic monomer is 1: 10-100.
6. The method for preparing the sensitive western blotting material based on the block macromolecular chain monomer according to claim 4, characterized in that: in the step 2, the molar ratio of the block macromolecular chain precursor 1 to the hydroxyethyl acrylate is 1: 10-100.
7. The method for preparing the sensitive western blotting material based on the block macromolecular chain monomer according to claim 1, characterized in that: the molar ratio of the block copolymerization macromolecular chain precursor 2 to the acryloyl chloride in the step 3 is 1: 10-100.
8. The method for preparing the sensitive western blotting material based on the block macromolecular chain monomer according to claim 1, characterized in that: the mass ratio of the protein to the macromolecular functional monomer in the step 4 is 1: 1-10.
9. The method for preparing the sensitive western blotting material based on the block macromolecular chain monomer according to claim 1, characterized in that: the organic solvent in the step 1 is N, N-dimethyl sulfoxide; the initiator is azobisisobutyronitrile.
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