Amino acid nano hydrogel and preparation method and application thereof
阅读说明：本技术 氨基酸纳米水凝胶及其制备方法和应用 (Amino acid nano hydrogel and preparation method and application thereof ) 是由 朱全红 朱泳妍 吴登宇 于 2019-09-06 设计创作，主要内容包括：本发明公开了一种氨基酸纳米水凝胶及其制备方法和应用,所述的氨基酸纳米水凝胶,具有氨基酸侧链,可以与蛋白质之间发生类似天然蛋白-天然蛋白的相互作用,对人血清蛋白及生物标志物β-淀粉样蛋白、脑钠肽、胃泌素释放肽前体等均具有特异性识别能力,可用于吸附目标蛋白。本发明提高了聚合物的识别能力,制备方法简单,反应条件温和,具有温度响应特性,具有发展为一种性能稳定的人工模拟抗体或蛋白质提纯高分子材料的潜力。所述的氨基酸纳米水凝胶,为聚合物A或聚合物B；所述聚合物A由式(Ⅰ)、式(Ⅱ)和式(Ⅲ)所示的片段构成；所述聚合物B由式(Ⅳ)、式(Ⅱ)、式(Ⅴ)和式(Ⅲ)所示的片段构成；<Image he="279" wi="700" file="DDA0002194002700000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention discloses an amino acid nano hydrogel and a preparation method and application thereof, wherein the amino acid nano hydrogel has an amino acid side chain, can generate interaction similar to natural protein-natural protein with protein, and is used for human serum proteinAnd biomarkers beta-amyloid, brain natriuretic peptide, gastrin-releasing peptide precursor, and the like, all have specific recognition capability, and can be used for adsorbing target proteins. The invention improves the identification capability of the polymer, has simple preparation method, mild reaction conditions and temperature response characteristic, and has the potential of developing an artificial simulated antibody with stable performance or purifying a high molecular material by protein. The amino acid nano hydrogel is a polymer A or a polymer B; the polymer A is composed of segments shown in a formula (I), a formula (II) and a formula (III); the polymer B is composed of segments shown in a formula (IV), a formula (II), a formula (V) and a formula (III);)
1. The amino acid nano hydrogel is characterized by being a polymer A or a polymer B;
the polymer A is composed of segments shown in a formula (I), a formula (II) and a formula (III);
the polymer B is composed of segments shown in a formula (IV), a formula (II), a formula (V) and a formula (III);
wherein: r1Represents C6H5Or CH (CH)3)2；R2Is represented by (CH)2)2CN3H5 +Or C3N2H3。
2. The amino acid nano hydrogel according to claim 1, wherein the number average molecular weight of the polymer A is 20kDa to 50kDa, and the average particle size is 100 nm to 150 nm; the critical phase transition temperature is 30-32 ℃.
3. The amino acid nano hydrogel according to claim 1, wherein the number average molecular weight of the polymer B is 20kDa to 50kDa, the average particle size is 60 nm to 75nm, and the critical phase transition temperature is 13 ℃ to 14 ℃.
4. The method for preparing the amino acid nano hydrogel according to claim 1, wherein the method for preparing the polymer A comprises the following steps:
s1, dissolving an N-acryloyl-L-hydrophobic amino acid functional monomer, a functional monomer N-isopropyl acrylamide, a cross-linking agent and an initiator in an anionic surfactant aqueous solution to obtain a reaction solution;
s2, reacting the reaction solution obtained in the step S1 in an inert atmosphere to obtain a polymerization solution, and collecting the polymer A from the reaction system.
5. The method of claim 4, wherein the N-acryloyl-L-hydrophobic amino acid functional monomer is N-acryloyl-L-phenylalanine or N-acryloyl-L-leucine;
the cross-linking agent is selected from more than one of N, N' -methylene bisacrylamide, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate or divinylbenzene;
the initiator is selected from more than one of ammonium persulfate, sodium persulfate, azobisisobutyronitrile or dibenzoyl peroxide.
6. The method according to claim 5, wherein the mole percentages of the components are:
2-47% of N-acryloyl-L-hydrophobic amino acid functional monomer;
50-97% of functional monomer N-isopropyl acrylamide;
1-3% of a cross-linking agent;
wherein the sum of the mole percentages of the monomers and the cross-linking agent is 100 percent;
the mass percentage concentration of the initiator in the reaction liquid is 0.01-0.1%;
the mass percentage concentration of the surfactant in the reaction liquid is 0.01-0.1%, and the total mole number of the N-acryloyl-L-hydrophobic amino acid functional monomer, the functional monomer N-isopropyl acrylamide and the cross-linking agent in the reaction liquid is 50-70 mmol/L.
7. The method for preparing the amino acid nano hydrogel according to claim 1, wherein the method for preparing the polymer B comprises the following steps:
p1, dissolving an N-acryloyl-L-amino acid functional monomer with positive charge, a functional monomer N-isopropyl acrylamide and a cross-linking agent in an aqueous solution containing a cationic surfactant to obtain an aqueous solution A;
dissolving a functional monomer N-tert-butyl acrylamide in an alcohol solvent, dissolving an initiator in a ketone solvent, and then mixing with an aqueous solution A to obtain a reaction solution;
and P2, reacting the reaction solution obtained in the step P1 in an inert atmosphere to obtain a polymerization solution, and collecting the polymer B from the reaction system.
8. The method for preparing amino acid nano hydrogel according to claim 7, wherein the N-acryloyl-L-positively charged amino acid functional monomer is N-acryloyl-L-arginine or N-acryloyl-L-histidine;
the cross-linking agent is selected from one or more of N, N' -methylene bisacrylamide, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate or divinylbenzene;
the cationic surfactant is selected from one or more of cetyl trimethyl ammonium bromide, octadecyl trimethyl ammonium chloride, dodecyl trimethyl ammonium chloride or dodecyl dimethyl benzyl ammonium chloride.
9. The method for preparing the amino acid nano hydrogel according to claim 7, wherein the mol percentages of the components are as follows:
in the aqueous solution containing the cationic surfactant, the mass percent concentration of the cationic surfactant is 0.01-0.1%;
in the aqueous solution A, the total mole number of the N-acryloyl-L-amino acid functional monomer with positive charges, the functional monomer N-isopropyl acrylamide, the functional monomer N-tert-butyl acrylamide and the cross-linking agent is 50-70 millimoles/L;
in the alcohol solvent, the mole number of the functional monomer N-tert-butyl acrylamide is 1.0-1.3 mmol/ml;
in the ketone solvent, the mass concentration of the initiator is 40-80 mg/ml;
the volume ratio of the alcohol solvent containing the functional monomer N-tertiary butyl acrylamide to the ketone solvent containing the initiator to the aqueous solution A is 2-3: 1-2: 100.
10. The use of the amino acid nanohydrogel of any one of claims 1 to 3 for adsorbing proteins, which are human serum proteins and the biomarkers β -amyloid, brain natriuretic peptide or gastrin releasing peptide precursor.
Technical Field
The invention relates to the technical field of non-biological antibodies, in particular to an amino acid nano hydrogel and a preparation method and application thereof.
Background
Antibodies play important roles in clinical detection, diagnosis and treatment of diseases, purification of antigens and proteins, and the like. The artificially prepared natural antibody is mainly obtained by methods such as a polyclonal antibody, a monoclonal antibody, a genetic engineering antibody and the like, wherein the monoclonal antibody is most commonly applied in clinical diagnosis and detection, but has long preparation period and high cost, is difficult to produce in a large scale, is expensive and cannot be recycled; on the other hand, the antibody is as sensitive to the environment as protein, has poor stability, and is easy to inactivate under high temperature and severe conditions, which brings great inconvenience to storage, transportation and application. Therefore, researchers are actively working on finding non-biological antibodies that are simple to prepare, stable in performance, and comparable in specificity to natural antibodies.
Molecular imprinting is a technique developed on the basis of simulation of natural antigen-antibody, enzyme-substrate interaction and biomimetic science, and can introduce molecular recognition holes and sites matching with specific target proteins in space and binding sites into polymers, thereby specifically recognizing the target proteins. For example, patent application with publication number CN 1911975a discloses a protein-imprinted amphiphilic polymer and a preparation method thereof, wherein the polymer is prepared by copolymerizing lysozyme and bovine serum albumin as template molecules and N- [3- (dimethylamino) propyl ] methacrylamide and methacrylic acid as functional monomers, and has imprinted cavities after being eluted by a template, and tertiary amine and carboxyl groups on the polymer can generate electrostatic interaction with groups on the template protein, so that the polymer has specific recognition capability on the template protein lysozyme and the bovine serum albumin. The patent with the publication number of CN 102532408B relates to a preparation method of a temperature-sensitive magnetic western-blot nanosphere, which not only has a blot cavity matched with template protein, but also has the characteristics of temperature sensitivity and rapid separation under an external magnetic field, and can be used for removing or enriching high-abundance protein components. The patent application with the publication number of CN101173028A discloses a preparation method and application of a protein imprinted resin with a cation recognition group, wherein a cloned porcine cyclophilin 18 protein is adopted as a template protein, so that the bottleneck that natural trace protein is adopted as a template molecule is overcome, and meanwhile, resin balling and a molecular imprinting technology are carried out step by step, so that the damage of the template molecule in the elution process can be reduced. The patent application with the publication number of CN 104941602A discloses a protein epitope molecularly imprinted material, and preparation and application thereof, wherein the molecularly imprinted material adopts a section of specific peptide section corresponding to target protein as template molecule, and can overcome the bottleneck of micro quality of natural protein. However, in the above-mentioned preparation of polymers with imprinted pores by using molecular imprinting technology, template protein, cloned protein or specific peptide fragment must be used as template molecules in the preparation process, and these template protein molecules, which are expensive and have unstable physicochemical properties, are eluted by using acidic and polar solvents during the imprinting process to cause structural denaturation, which leads to non-reuse; and the template elution often has the phenomenon of incomplete elution, which can cause the nonuniformity of imprinting holes, influence the adsorption of the polymer, even cause the phenomenon of leakage when the polymer adsorbs target protein, and cause the problems of inaccurate adsorption quantity and the like. In addition, the protein has a complex structure and a plurality of action sites, and the prepared polymer three-dimensional network structure is unstable, so that the collapse of a recognition site is easily caused.
The nano polymer is a new direction for preparing the non-biological antibody. It is a high molecular polymer formed by the interaction of functional monomer, initiator and cross-linking agent without adding template molecule in the preparation process.
Research results of scholars show that functional monomers play a key role in the process of polymer identification besides imprinting holes, so that the development and research of a novel functional monomer and the nano-polymer obtained by polymerizing the functional monomer can overcome the defects in the prior art, and the method is very desirable.
Disclosure of Invention
The invention provides the amino acid nano hydrogel for overcoming the defects in the prior art, template molecules are not added in the preparation process of the amino acid nano hydrogel, template elution is not needed, the structure is stable, the temperature responsiveness is presented, the amino acid side chain can generate the interaction of natural protein and natural protein with protein, and the amino acid nano hydrogel has the specific recognition capability on human serum protein.
The invention also aims to provide a preparation method of the amino acid nano hydrogel.
The invention also aims to provide the application of the amino acid nano hydrogel in protein adsorption.
In order to solve the technical problems, the invention adopts the technical scheme that:
the amino acid nano hydrogel is a polymer A or a polymer B;
the polymer A is composed of segments shown in a formula (I), a formula (II) and a formula (III);
the polymer B is composed of segments shown in a formula (IV), a formula (II), a formula (V) and a formula (III);
wherein: r1Represents C6H5Or CH (CH)3)2；R2Is represented by (CH)2)2CN3H5 +Or C3N2H3。
C above6H5Represents a phenyl group. CH (CH) as described above3) Represents an isopropyl group.
Above (CH)2)2CN3H5 +To represent
Preferably, the number average molecular weight of the polymer A is 20 kDa-50 kDa, and the average particle size is 100-150 nm; the critical phase transition temperature is 30-32 ℃.
Preferably, the number average molecular weight of the polymer B is 20-50 kDa, the average particle size is 60-75 nm, and the critical phase transition temperature is 13-14 ℃.
The polymer A is hydrophobic amino acid nano hydrogel. The polymer B is amino acid nano hydrogel with positive charges.
The preparation method of the polymer A comprises the following steps:
s1, dissolving an N-acryloyl-L-hydrophobic amino acid functional monomer, a functional monomer N-isopropyl acrylamide, a cross-linking agent and an initiator in an anionic surfactant aqueous solution to obtain a reaction solution;
s2, reacting the reaction solution obtained in the step S1 in an inert atmosphere to obtain a polymerization solution, and collecting the polymer A from the reaction system.
And S2, the inert atmosphere can be a nitrogen atmosphere. And S2, the reaction temperature is 50-70 ℃, and the reaction temperature is 3-6 h.
Preferably, the collection method in step s2. is a dialysis method. The method specifically comprises the following steps:
putting the obtained polymer liquid into a dialysis bag (MW 12000-14000) for dialysis for 2-6 days, preferably 4 days, and changing water 1-3 times per day, preferably 2 times;
and collecting the dialysate inside the dialysis membrane, and freeze-drying to obtain the polymer A which is the hydrophobic amino acid nano hydrogel.
Preferably, the N-acryloyl-L-hydrophobic amino acid functional monomer is N-acryloyl-L-phenylalanine or N-acryloyl-L-leucine.
Preferably, the anionic surfactant is selected from one or more of sodium lauryl sulfate, sodium lauryl sulfonate, sodium stearate or sodium disproportionate abietate.
Preferably, the crosslinking agent in step s1. is selected from one or more of N, N' -methylenebisacrylamide, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, or divinylbenzene.
Preferably, the initiator in step s1. is selected from more than one of ammonium persulfate, sodium persulfate, azobisisobutyronitrile or dibenzoyl peroxide.
Preferably, the mole percentages of the components are as follows:
2-47% of N-acryloyl-L-hydrophobic amino acid functional monomer;
50-97% of functional monomer N-isopropyl acrylamide;
1-3% of a cross-linking agent;
wherein the sum of the mole percentages of the monomers and the cross-linking agent is 100 percent;
s1, the mass percentage concentration of the initiator in the reaction liquid is 0.01-0.1%;
the mass percentage concentration of the surfactant in the reaction liquid is 0.01-0.1%, and the total mole number of the N-acryloyl-L-hydrophobic amino acid functional monomer, the functional monomer N-isopropyl acrylamide and the cross-linking agent in the reaction liquid is 50-70 mmol/L.
The preparation method of the polymer B comprises the following steps:
p1, dissolving an N-acryloyl-L-amino acid functional monomer with positive charge, a functional monomer N-isopropyl acrylamide and a cross-linking agent in an aqueous solution containing a cationic surfactant to obtain an aqueous solution A;
dissolving a functional monomer N-tert-butyl acrylamide in an alcohol solvent, dissolving an initiator in a ketone solvent, and then mixing with an aqueous solution A to obtain a reaction solution;
and P2, reacting the reaction solution obtained in the step P1 in an inert atmosphere to obtain a polymerization solution, and collecting the polymer B from the reaction system.
Step p2. the inert atmosphere may be a nitrogen atmosphere. And S2, the reaction temperature is 50-70 ℃, and the reaction temperature is 3-6 h.
Preferably, the collection method in step p2. is a dialysis method. The method specifically comprises the following steps:
putting the obtained polymer liquid into a dialysis bag (MW 12000-14000) for dialysis for 2-6 days, preferably 4 days, and changing water 1-3 times per day, preferably 2 times;
and collecting dialysate inside the dialysis membrane, and freeze-drying to obtain the polymer B which is the amino acid nano hydrogel with positive charges.
Preferably, the N-acryloyl-L-positively charged amino acid functional monomer is N-acryloyl-L-arginine or N-acryloyl-L-histidine.
Preferably, the crosslinking agent described in step p1. is selected from one or more of N, N' -methylenebisacrylamide, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate or divinylbenzene.
Preferably, the initiator in step p1. is selected from one or more of ammonium persulfate, sodium persulfate, azobisisobutyronitrile or dibenzoyl peroxide.
Preferably, the cationic surfactant is selected from one or more of cetyl trimethyl ammonium bromide, stearyl trimethyl ammonium chloride, dodecyl trimethyl ammonium chloride or dodecyl dimethyl benzyl ammonium chloride.
The alcohol solvent is preferably ethanol, methanol, isopropanol or n-butanol.
The ketone solvent is preferably acetone, butanone, acetylacetone or chloropropanone.
Preferably, the mole percentages of the components are as follows:
wherein the sum of the mole percentages of the monomers and the cross-linking agent is 100 percent;
in the aqueous solution containing the cationic surfactant, the mass percent concentration of the cationic surfactant is 0.01-0.1%;
in the aqueous solution A, the total mole number of the N-acryloyl-L-amino acid functional monomer with positive charges, the functional monomer N-isopropyl acrylamide, the functional monomer N-tert-butyl acrylamide and the cross-linking agent is 50-70 millimoles/L;
in the alcohol solvent, the mole number of the functional monomer N-tert-butyl acrylamide is 1.0-1.3 mmol/ml;
in the ketone solvent, the mass concentration of the initiator is 40-80 mg/ml;
the volume ratio of the alcohol solvent containing the functional monomer N-tertiary butyl acrylamide to the ketone solvent containing the initiator to the aqueous solution A is 2-3: 1-2: 100.
The invention also protects the application of the amino acid nano hydrogel in protein adsorption, wherein the protein is human serum protein and a biomarker beta-amyloid protein, brain natriuretic peptide or gastrin release peptide precursor.
The amino acid nano hydrogel has an amino acid side chain, can interact with proteins similar to natural protein-natural protein, has specific recognition capability on human serum protein and the like, has specificity on the human serum protein and the like, is obviously superior to other three kinds of amino acid nano hydrogels, can be used for adsorbing target proteins such as human serum protein and biomarker beta-amyloid, brain natriuretic peptide, gastrin release peptide precursor and the like, and has the potential of developing into an artificial simulated antibody with stable performance or a protein purified high molecular material.
Compared with the prior art, the invention has the beneficial effects that:
by improving the functional monomer, the recognition capability of the polymer is improved, and the obtained nano polymer has an amino acid side chain and can perform interaction similar to natural protein-natural protein with target protein. Meanwhile, in order to improve the biocompatibility of the polymer and exert the performance similar to a natural antibody, the nano polymer can be prepared into nano hydrogel: (1) the particle size is usually 1-1000 nm, the specific surface area is large, and functional groups on the surface can be coupled with components with specific functions and can instantaneously respond to stimuli in a microenvironment, such as temperature, light, pH value and the like; (2) the stability is good, and the internal cross-linked network structure makes the nano-micelle and nano-vesicle more stable than the nano-micelle and nano-vesicle; (3) has high water content, is similar to biological tissues, and has good biocompatibility.
The preparation method is simple, the reaction condition is mild, and the preparation method can be carried out in aqueous solution; the temperature-sensitive N-isopropylacrylamide is adopted as a functional monomer, so that the obtained four amino acid nano hydrogel has temperature responsiveness, APA @ NPs and A-Leu @ NPs have low critical phase transition temperature (LCST) values, A-Arg @ NPs and A-His @ NPs have high critical phase transition temperature (UCST) values, and the amino acid side chains of the hydrogel can interact with protein through natural protein-natural protein, so that the hydrogel has specific recognition capability on human serum protein, especially the specificity of arginine nano polymer on the human serum protein, and has the potential of developing into an artificial simulated antibody with stable performance or a protein purification high molecular material.
Drawings
FIG. 1 shows the particle size of the amino acid nano-hydrogel of example 1 and example 2 of the present invention as a function of temperature.
FIG. 2 is the NMR spectrum of the phenylalanine nano-hydrogel of example 1 of the present invention.
FIG. 3 is the NMR spectrum of the leucine nano-hydrogel of example 1 of the present invention.
FIG. 4 is the NMR spectrum of the arginine nano-hydrogel of example 2 of the present invention.
FIG. 5 is the NMR spectrum of histidine nano-hydrogel in example 2 of the present invention.
Fig. 6 shows the binding rates of the amino acid nano-hydrogels of example 1 and example 2 of the present invention to five proteins.
FIG. 7 is MST trace thermophoresis images of phenylalanine nano-hydrogel to human serum protein, ovalbumin and alpha-lactalbumin in example 1 of the present invention. In FIG. 7, scale values on the abscissa axis are
Detailed Description
The present invention will be further described with reference to the following embodiments.
The same or similar reference numerals in the drawings of the embodiments of the present invention correspond to the same or similar components; in the description of the present invention, it is to be understood that the terms "upper", "lower", "left", "right", "top", "bottom", "inner", "outer", and the like, if any, are used in the orientations and positional relationships indicated in the drawings only for the convenience of describing the present invention and simplifying the description, but not for indicating or implying that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and therefore the terms describing the positional relationships in the drawings are used for illustrative purposes only and are not to be construed as limiting the present patent.
Furthermore, if the terms "first," "second," and the like are used for descriptive purposes only, they are used for mainly distinguishing different devices, elements or components (the specific types and configurations may be the same or different), and they are not used for indicating or implying relative importance or quantity among the devices, elements or components, but are not to be construed as indicating or implying relative importance.