CN114395135B - Magnetic microsphere with protein non-specific adsorption resistance and preparation method thereof - Google Patents
Magnetic microsphere with protein non-specific adsorption resistance and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a magnetic microsphere with protein non-specific adsorption resistance and a preparation method thereof. The magnetic microsphere of the invention can resist protein nonspecific adsorption and can be coupled with other biomolecules, thereby being capable of specifically adsorbing target protein in a complex environment and greatly improving the detection sensitivity.
Description
Technical Field
The invention relates to a magnetic microsphere with protein non-specific adsorption resistance and a preparation method thereof.
Background
The magnetic nanoparticles have the advantages of superparamagnetism, huge specific surface area, controllable size, good dispersibility and high stability, and are rapidly developed in the biomedical fields of nuclear magnetic resonance, protein purification, drug delivery, bacterial detection, catalysis and the like. Magnetic nanoparticles must have sufficient stability and good biocompatibility to be used in biomedical applications. However, the conventional magnetic nanospheres are prone to non-specific adsorption with a large amount of proteins in a complex biological environment, so that the detection sensitivity is reduced, and the magnetic nanospheres are also aggregated or precipitated, so that the functional modification of the magnetic nanospheres, the improvement of the water dispersibility of the magnetic nanospheres, the improvement of the stability and the biocompatibility, the improvement of the non-specific adsorption capacity of the proteins and the increase of the reactive sites are increasingly urgent.
The protein non-specific adsorption materials are mainly divided into two categories, namely hydrophilic non-zwitterionic polymers and zwitterionic polymers. The protein non-specific adsorption material of the non-zwitterionic polymer comprises polyethylene glycol and derivatives thereof, polysaccharide, polyamide and the like, which are good hydrophilic substances, are neutral in electricity and contain hydrogen receptors. Zwitterionic materials are mainly polybetaines and polyampholytes. Since 1970, there are successive researches on modifying the surface of the nano material by pegylation to increase the hydrophilicity and colloidal stability of the nano particles, and the nano particles are applied to the aspects of image diagnosis, disease treatment and the like.
Disclosure of Invention
The purpose of the invention is as follows: one of the purposes of the invention is to provide a magnetic microsphere which can resist the nonspecific adsorption of protein and can also utilize a surface active site to be chemically coupled with an antibody; the invention also aims to provide a preparation method of the magnetic microsphere.
The technical scheme is as follows: the magnetic microsphere with the function of resisting protein nonspecific adsorption has the chemical structural formula as follows:
wherein R is magnetic nanoparticles, and R' is hyperbranched polyglycidyl ether.
Hyperbranched polyglycidyl ether has a hydrophilic polymer material with a highly branched structure, the branched structure can effectively prevent the surface of magnetic nanoparticles from contacting with protein in the environment, the adsorption is reduced by effectively isolating the surface of the material from non-specific adsorbed protein, in addition, the tail end of the hyperbranched polyglycidyl ether molecule has a large number of hydroxyl functional groups, a large number of ether bonds are also contained in the molecule, the hyperbranched polyglycidyl ether molecule and the water molecule can form hydrogen bonds, the hyperbranched polyglycidyl ether molecule and the water molecule are combined into a hydration layer through the action of the hydrogen bonds to reduce the adsorption, and meanwhile, the hyperbranched polyglycidyl ether has high solubility in water.
The preparation method of the magnetic microsphere with the function of resisting the protein non-specific adsorption comprises the following steps: firstly, diphenylmethane diisocyanate is taken as a bridging intermediate to modify hyperbranched polyglycidyl ether on the surface of carboxylated magnetic nanoparticles, and the grafted hyperbranched polyglycidyl ether is reacted with p-toluenesulfonyl chloride to generate the magnetic microspheres with sulfonic ester active groups.
The carboxylated magnetic microsphere prepared by the reflux precipitation method is firstly grafted with hyperbranched polyglycidyl ether on the surface and then reacts with p-methylbenzenesulfonyl chloride, and the surface of the magnetic microsphere not only has hyperbranched polyglycidyl ether which can effectively resist protein non-specific adsorption components, but also has active sites which can be coupled with other biomolecules.
Wherein, the carboxylated magnetic nano-particles are carboxylated ferroferric oxide nano-particles.
Wherein, the diphenylmethane diisocyanate is used as a bridging intermediate to connect the carboxylated ferroferric oxide and the hyperbranched polyglycidyl ether, and the mass ratio of the diphenylmethane diisocyanate to the carboxylated ferroferric oxide is 2.5-4: 1.
wherein the mass ratio of the hyperbranched polyglycidyl ether to the carboxylated ferroferric oxide is 2-3: 1.
wherein the mass ratio of the carboxylated ferroferric oxide to the p-methylbenzenesulfonyl chloride is 1-2: 1.
wherein, in the reaction process of the carboxylated ferroferric oxide and the diphenylmethane diisocyanate, the reaction temperature is 80-90 ℃ and the reaction time is 12-24 hours.
Wherein, in the reaction process with the hyperbranched polyglycidyl ether, the reaction temperature is 80-90 ℃ and the reaction time is 12-24 h.
Wherein, in the reaction process with the p-toluenesulfonyl chloride, sodium hydroxide solution is also added, the sodium hydroxide is used as a catalyst, the reaction is kept under an alkaline environment, and the concentration of the sodium hydroxide solution is 0.25-0.3M.
Wherein, in the reaction process with the p-methyl benzene sulfonyl chloride, the reaction temperature is 0-1 ℃, and the reaction time is 12-24 h.
The chemical reaction equation of the magnetic microsphere is as follows:
the application of the magnetic microsphere of the invention is as follows:
R 1 is an antibody molecule.
The magnetic microsphere obtained by the method is grafted with a specific antibody at 37 ℃, the amino group of an antibody molecule replaces the active group sulfonate of the magnetic microsphere, the sulfonate is unstable, the amino group and the hyperbranched polyglycidyl ether generate more stable groups, and after the magnetic microsphere is grafted with the specific antibody molecule, the antigen corresponding to the antibody is detected, so that the detection of the antigen content is realized.
Has the advantages that: the hyperbranched polyglycidyl ether is grafted on the carboxylated magnetic microspheres firstly, so that the nonspecific adsorption of the magnetic microspheres to protein is effectively reduced, then the p-toluenesulfonyl chloride reacts with the hydroxyl on the hyperbranched polyglycidyl ether, so that the surfaces of the magnetic microspheres contain active sites, the magnetic microspheres which can resist the nonspecific adsorption of protein and can be coupled with other biomolecules are obtained, and the final product can specifically adsorb target protein in a complex environment, so that the detection sensitivity is greatly improved, and the application prospect in the biological aspect is great.
Drawings
FIG. 1 is a transmission electron microscope image of carboxylated magnetic microspheres prepared by a reflux precipitation method;
FIG. 2 is a transmission electron micrograph of the magnetic microsphere with anti-nonspecific adsorption of proteins prepared in example 1;
FIG. 3 is a standard curve for the measurement of bovine serum albumin concentration in magnetic microspheres by Coomassie Brilliant blue (Bradford);
FIG. 4 is a graph showing the ultraviolet contrast of supernatant fluid obtained from a nonspecific adsorption experiment of bovine serum albumin before and after grafting of carboxylated magnetic microspheres with hyperbranched polyglycidyl ether;
FIG. 5 shows the calculation of the specific nonspecific adsorption values before and after grafting from the UV data.
Detailed Description
Example 1
The preparation method of the magnetic microsphere for resisting protein non-specific adsorption comprises the following steps:
(1) 0.1g of carboxylated magnetic microspheres Fe are weighed 3 O 4 @ MAA was dispersed in 20ml DMF solvent (N, N-dimethylformamide solution), sonicated for 10min, and 0.25g MDI (diphenylmethane diisocyanate) was added theretoNitrile acid ester), continuing to perform ultrasonic treatment for 10min to form a uniform solution, reacting at 80 ℃ for 12h, and performing magnetic separation after the reaction is finished;
(2) Magnetically separating the product obtained in the step (1), dispersing the product into a 20ml DMF solvent again, adding 0.3PGH (hyperbranched polyglycidyl ether) to react at 80 ℃ for 12 hours, and magnetically separating again after the reaction is finished;
(3) Magnetically separating the product of the step (2), dispersing the product into a 20mLDMF solvent again, adding 0.1g of Tosyl (p-toluenesulfonyl chloride) and 20mL of NaOH aqueous solution with the concentration of 0.25M, reacting at 0 ℃ for 12h, and reacting the product Fe after reaction 3 O 4 @ MAA-HPG-Tosyl is washed twice with ethanol, three times with distilled water, and dispersed in distilled water for use.
Example 2
The preparation method of the magnetic microsphere with the protein non-specific adsorption resistance comprises the following steps:
(1) 0.1g of carboxylated magnetic microspheres Fe are weighed 3 O 4 @ MAA is dispersed in 30ml DMF solvent, ultrasonic treatment is carried out for 10min, 0.3g of MDI is added into the solvent, the ultrasonic treatment is continued for 10min to form uniform solution, the reaction is carried out for 24h at 80 ℃, and magnetic separation is carried out after the reaction is finished;
(2) Magnetically separating the product obtained in the step (1), dispersing the product into a 40mLDMF solvent again, adding 0.3PGH, reacting at 80 ℃ for 24 hours, and magnetically separating again after the reaction is finished;
(3) Magnetically separating the product of the step (2), dispersing into 40mL of mixed solution of sodium hydroxide and sodium hydroxide again, adding 0.1g of Tosyl and 40mL of NaOH aqueous solution with the concentration of 0.3M, reacting at 0 ℃ for 24h, and reacting the product Fe after reaction 3 O 4 @ MAA-HPG-Tosyl is washed twice with ethanol, three times with distilled water, and dispersed in distilled water for use.
Example 3
The preparation method of the magnetic microsphere for resisting protein non-specific adsorption comprises the following steps:
(1) 0.1g of carboxylated magnetic microspheres Fe are weighed 3 O 4 @ MAA is dispersed in 40ml DMF solvent, ultrasonic treatment is carried out for 10min, 0.4g of MDI is added into the solvent, the ultrasonic treatment is continued for 10min to form uniform solution, the temperature is increased to 90 ℃ for reaction for 24h, and magnetic separation is carried out after the reaction is finished;
(2) Magnetically separating the product obtained in the step (1), dispersing the product into 40ml of DMF solvent again, adding 0.3PGH, reacting for 24 hours at 80 ℃, and magnetically separating again after the reaction is finished;
(3) Magnetically separating the product of the step (2), dispersing into 40mL of mixed solution of sodium hydroxide and sodium hydroxide again, adding 0.1g of Tosyl and 40mL of NaOH aqueous solution with the concentration of 0.3M, reacting at 0 ℃ for 24h, and reacting the product Fe after reaction 3 O 4 @ MAA-HPG-Tosyl is washed twice with ethanol, three times with distilled water, and dispersed in distilled water for use.
And (3) performing a protein non-specific adsorption resistance test on the magnetic microspheres: protein concentration is measured by a Bradford method (Coomassie brilliant blue method), a standard curve as shown in figure 3 is obtained, and the nonspecific adsorption of the modified magnetic microspheres is reduced to 46.85% of the original nonspecific adsorption through calculation results, which indicates that the prepared magnetic microspheres have the property of resisting the nonspecific adsorption of protein. The standard curve is that bovine serum albumin ultraviolet absorption peak intensity of different concentrations is linear increase, incubate with bovine serum albumin and magnetic bead, survey the ultraviolet absorption intensity of supernatant after the magnetic separation, correspond the standard curve, can obtain supernatant bovine serum albumin concentration this moment to calculate the nonspecific adsorption capacity of magnetic bead. Compared with the non-specific adsorption of the modified magnetic microspheres to the protein before modification, the non-specific adsorption of the modified magnetic microspheres to the protein is greatly reduced, so that the background value can be reduced on the application prospect of the specific binding antigen, and the detection sensitivity is improved.
Claims (10)
1. A magnetic microsphere with protein nonspecific adsorption resistance is characterized in that the chemical structural formula of the magnetic microsphere is as follows:
wherein R is magnetic nanoparticles, and R' is hyperbranched polyglycidyl ether.
2. The method for preparing magnetic microspheres resistant to protein non-specific adsorption according to claim 1, wherein the method comprises: firstly, diphenylmethane diisocyanate is used as a bridging intermediate to modify hyperbranched polyglycidyl ether on the surface of the carboxylated magnetic nanoparticles, and the grafted hyperbranched polyglycidyl ether reacts with p-methylbenzenesulfonyl chloride to generate the magnetic microspheres with sulfonic ester active groups.
3. The method for preparing magnetic microspheres resistant to protein non-specific adsorption according to claim 2, wherein: the carboxylated magnetic nano-particles are carboxylated ferroferric oxide nano-particles.
4. The method for preparing magnetic microspheres resistant to protein non-specific adsorption according to claim 2, wherein: diphenylmethane diisocyanate is used as a bridging intermediate to connect carboxylated ferroferric oxide and hyperbranched polyglycidyl ether, and the mass ratio of the diphenylmethane diisocyanate to the carboxylated ferroferric oxide is (2.5-4): 1.
5. the method for preparing magnetic microspheres resistant to protein non-specific adsorption according to claim 4, wherein: the mass ratio of the hyperbranched polyglycidyl ether to the carboxylated ferroferric oxide is 2-3: 1.
6. the method for preparing magnetic microspheres resistant to protein non-specific adsorption according to claim 4, wherein: in the reaction process of the carboxylated ferroferric oxide and the diphenylmethane diisocyanate, the reaction temperature is 80-90 ℃ and the reaction time is 12-24 hours.
7. The method for preparing magnetic microspheres resistant to protein non-specific adsorption according to claim 4, wherein: in the reaction process with the hyperbranched polyglycidyl ether, the reaction temperature is 80-90 ℃, and the reaction time is 12-24 h.
8. The method for preparing magnetic microspheres resistant to protein non-specific adsorption according to claim 2, wherein: the mass ratio of the carboxylated ferroferric oxide to the p-methylbenzenesulfonyl chloride is 1-2: 1.
9. the method for preparing magnetic microspheres resistant to protein non-specific adsorption according to claim 8, wherein: in the reaction process of the p-toluenesulfonyl chloride, sodium hydroxide solution is also added, and the concentration of the sodium hydroxide solution is 0.25-0.3M.
10. The method for preparing magnetic microspheres resistant to protein non-specific adsorption according to claim 8, wherein: in the reaction process with the p-methyl benzene sulfonyl chloride, the reaction temperature is 0-1 ℃, and the reaction time is 12-24 h.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007254650A (en) * | 2006-03-24 | 2007-10-04 | Fujifilm Corp | Hardened film, antireflection film, polarizing plate using them, and image display device |
| CN103446964A (en) * | 2013-08-29 | 2013-12-18 | 西北工业大学 | Preparation method of carboxyl functionalized hyperbranched magnetic mesoporous silica composite microspheres |
| CN104316679A (en) * | 2014-10-29 | 2015-01-28 | 南京基蛋生物科技有限公司 | Application of hyperbranched polyglycerol modified magnetic nanoparticle microspheres in chemiluminescence immune assay |
| CN112108128A (en) * | 2020-08-31 | 2020-12-22 | 西安交通大学 | Hydrophilic hyperbranched polyglycidyl ether anion magnetic adsorbent and preparation method and application thereof |
| CN113061601A (en) * | 2021-03-17 | 2021-07-02 | 东北农业大学 | Method for preparing immobilized phospholipase C based on multipoint covalent interaction |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2007254650A (en) * | 2006-03-24 | 2007-10-04 | Fujifilm Corp | Hardened film, antireflection film, polarizing plate using them, and image display device |
| CN103446964A (en) * | 2013-08-29 | 2013-12-18 | 西北工业大学 | Preparation method of carboxyl functionalized hyperbranched magnetic mesoporous silica composite microspheres |
| CN104316679A (en) * | 2014-10-29 | 2015-01-28 | 南京基蛋生物科技有限公司 | Application of hyperbranched polyglycerol modified magnetic nanoparticle microspheres in chemiluminescence immune assay |
| CN112108128A (en) * | 2020-08-31 | 2020-12-22 | 西安交通大学 | Hydrophilic hyperbranched polyglycidyl ether anion magnetic adsorbent and preparation method and application thereof |
| CN113061601A (en) * | 2021-03-17 | 2021-07-02 | 东北农业大学 | Method for preparing immobilized phospholipase C based on multipoint covalent interaction |
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