CN116593687A - Preparation method and application of protein-coupled nano magnetic beads - Google Patents
Preparation method and application of protein-coupled nano magnetic beads Download PDFInfo
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- CN116593687A CN116593687A CN202310414510.2A CN202310414510A CN116593687A CN 116593687 A CN116593687 A CN 116593687A CN 202310414510 A CN202310414510 A CN 202310414510A CN 116593687 A CN116593687 A CN 116593687A
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- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 7
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- 229940050906 magnesium chloride hexahydrate Drugs 0.000 claims description 4
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims description 4
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- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 11
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- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 238000003917 TEM image Methods 0.000 description 2
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
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- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method and application of protein-coupled nano magnetic beads, wherein the preparation method comprises the following steps: s1, washing polydopamine coated magnetic iron oxide particles, and dispersing the magnetic iron oxide particles in a first buffer solution to obtain a pretreatment material; s2, adding target protein for incubation treatment, and magnetically separating supernatant after incubation reaction to obtain protein-coupled nano magnetic beads; and S3, dispersing the protein-coupled nano magnetic beads in a second buffer solution for preservation. The preparation method of the protein-coupled nano magnetic beads has the advantages of low production cost, simple preparation steps, mild reaction conditions, short period and the like, and the prepared protein-coupled nano magnetic beads have good biological activity, high purification efficiency, long storage time and can be recycled.
Description
Technical Field
The invention relates to the field of nanotechnology and nanomedicine, in particular to a preparation method and application of protein-coupled nanomagnetic beads.
Background
In recent years, multifunctional nano magnetic beads modified by specific antibodies, proteins or receptors have become an important means for biomedical and scientific research, and have been widely used in various fields such as bioseparation, immunoassay, cell sorting, food safety detection, and enzyme immobilization. Functional modification of biological protein molecules is a very important technical link for producing high-quality nano magnetic bead products.
The two main means for combining the nanometer magnetic beads and the biological protein molecules are as follows: chemical coupling and physical adsorption. The chemical coupling method is to fix the biological molecule on the surface of the magnetic bead through covalent bond, so the chemical coupling has the advantages of good stability, long preservation time and the like, but in order to avoid the inactivation of protein molecules, the chemical coupling is usually carried out in aqueous solution under milder conditions, which leads to low coupling efficiency of the protein molecules. Although the physical adsorption method has the advantages of simple operation, mild condition and the like, the active protein molecules adsorbed on the surfaces of the magnetic beads are easy to fall off, the stability is poor, and the storage time is not long.
Therefore, under the condition of ensuring the activity of the molecules, it is important to develop a method for efficiently coupling protein molecules on the surface of magnetic beads.
Disclosure of Invention
The invention aims to solve one of the problems existing in the prior related art at least to a certain extent, and therefore, the invention provides a preparation method of the protein-coupled nano magnetic beads, which has the advantages of low production cost, simple preparation steps, mild reaction conditions, short period and the like, and the prepared protein-coupled nano magnetic beads have good biological activity, high purification efficiency, long storage time and can be recycled. The invention also provides application of the protein-coupled nano magnetic beads.
According to the preparation method of the protein-coupled nano magnetic beads, the preparation method is realized by the following technical scheme:
the preparation method of the protein-coupled nano magnetic beads comprises the following steps:
s1, washing polydopamine coated magnetic iron oxide particles, and dispersing the magnetic iron oxide particles in a first buffer solution to obtain a pretreatment material;
s2, adding target protein for incubation treatment, and magnetically separating supernatant after incubation reaction to obtain protein-coupled nano magnetic beads;
and S3, dispersing the protein-coupled nano magnetic beads in a second buffer solution for preservation.
Further, the first buffer solution comprises PBS; the specific steps of dispersing the polydopamine-coated magnetic iron oxide particles in the first buffer solution after washing comprise: after washing the polydopamine coated magnetic iron oxide particles at least once with ultrapure water, the polydopamine coated magnetic iron oxide particles are ultrasonically dispersed in PBS.
Further, the added target Protein comprises Protein L; the specific steps of adding target protein for incubation treatment comprise: taking a quantitative pretreatment material, magnetically separating supernatant, adding 2 XPBS, protein L and ultrapure water, uniformly mixing, and then placing into a shaking table for incubation reaction.
Further, the pH value of the 2 XPBS is 5.0-9.0, the input amount of the Protein L is 5-15% of the amount of the pretreatment material, the reaction temperature is 25+/-5 ℃, and the incubation reaction time is 6-24h.
Further, the second buffer comprises PBST comprising 1 x PBS and 0.5% tween-20; the preservation temperature is 4+/-1 ℃ and the concentration of the magnetic beads is 10mg/mL.
Further, the preparation of the polydopamine coated magnetic iron oxide particles specifically comprises the following steps:
s11, self-making magnetic iron oxide nano particles;
s12, performing magnetic separation and washing treatment on the magnetic iron oxide nano particles, and then dispersing the magnetic iron oxide nano particles in a solvent by ultrasonic to obtain a pretreated material;
and S13, adding dopamine for polymerization to obtain polydopamine-coated magnetic iron oxide particles.
Further, the magnetic ferric oxide nano particles are ferroferric oxide nano materials and/or metal ion doped ferroferric oxide nano materials, wherein the metal ions are any one of magnesium ions, cobalt ions, nickel ions or zinc ions; the input amount of the dopamine is 5-75% of the amount of the pretreatment material, and the polymerization reaction time is 6-24 hours.
Further, the specific steps of the self-made magnetic iron oxide nanoparticle include: mixing ferric chloride hexahydrate, magnesium chloride hexahydrate, anhydrous sodium acetate, ethylene glycol and polyethylene glycol 600, stirring uniformly, and then placing into a reaction kettle for high-temperature treatment.
Further, the specific steps of adding dopamine for polymerization comprise: washing a quantitative pretreatment material, magnetically separating a supernatant, adding a second buffer solution and dopamine, uniformly mixing by ultrasound, and then putting into a shaking table for oscillating reaction.
According to the application of the protein-coupled nano magnetic beads, the application is realized by the following technical scheme:
an application of protein-coupled nanomagnetic beads, which has the preparation method of the protein-coupled nanomagnetic beads, wherein the protein-coupled nanomagnetic beads are used for purifying IgG in a target object, and the target object is any one of mixed solution of fetal Bovine Serum (BSA) and IgG, mixed solution of cell lysate and IgG or human serum.
Compared with the prior art, the invention at least comprises the following beneficial effects:
1. compared with the traditional chemical coupling method, the preparation method of the protein-coupled nano magnetic beads provided by the invention is based on the condensation reaction between carbonyl in polydopamine and amino in protein, and has the advantages of mild reaction conditions, simple preparation steps, low production cost, short period, good repeatability, good biological activity, high purification efficiency, long preservation time and the like;
2. the protein coupling nano magnetic beads prepared by the method have strong universality and are basically suitable for coupling modification and functionalization of any one protein on the surfaces of the magnetic beads;
3. the prepared protein-coupled nano magnetic beads have higher purification efficiency and excellent selectivity on human immunoglobulin G, can specifically separate and purify the human immunoglobulin G in fetal calf serum, cell lysate or human serum, and can achieve the adsorption quantity of about 10% (W/W) on the human immunoglobulin G.
Drawings
FIG. 1 is a schematic diagram of Mg-Fe in an embodiment of the invention 3 O 4 Nanometer magnetic bead and Mg-Fe 3 O 4 SEM image of PDA nanomagnetic beads;
FIG. 2 is a schematic diagram of Mg-Fe in an embodiment of the invention 3 O 4 TEM image of PDA nano magnetic beads;
FIG. 3 is a schematic diagram of Mg-Fe in an embodiment of the invention 3 O 4 Nanometer magnetic bead and Mg-Fe 3 O 4 IR diagram of PDA nanomagnetic beads;
FIG. 4 is a schematic diagram of Mg-Fe in an embodiment of the invention 3 O 4 SDS-page diagram of purifying IgG by PDA-Protein L nano magnetic beads in mixed solution of BSA and IgG;
FIG. 5 is a schematic diagram of Mg-Fe in an embodiment of the invention 3 O 4 SDS-page diagram of purified IgG of PDA-Protein L nanometer magnetic beads in the mixture of cell lysate and IgG;
FIG. 6 is a schematic diagram of Mg-Fe in an embodiment of the invention 3 O 4 SDS-page of purified IgG in human serum with PDA-Protein L nanobeads.
Detailed Description
The following examples illustrate the invention, but the invention is not limited to these examples. Modifications and equivalents of some of the technical features of the specific embodiments of the present invention may be made without departing from the spirit of the present invention, and they are all included in the scope of the claimed invention.
The invention provides a preparation method of protein-coupled nano magnetic beads, which is suitable for coupling specific antibodies and other proteins on the surfaces of magnetic beads. The preparation method comprises the following steps:
s1, washing polydopamine coated magnetic iron oxide particles, and dispersing the magnetic iron oxide particles in a first buffer solution to obtain a pretreatment material;
specifically, the first buffer solution comprises PBS; and washing the polydopamine-coated magnetic iron oxide particles with ultrapure water at least once, and then dispersing the polydopamine-coated magnetic iron oxide particles in PBS (phosphate buffer solution) by ultrasonic waves to obtain the pretreatment material.
S2, adding target protein for incubation treatment, and magnetically separating supernatant after incubation reaction to obtain protein-coupled nano magnetic beads;
specifically, the target Protein includes Protein L. Taking a quantitative pretreatment material, magnetically separating supernatant, adding 2 XPBS, protein L and ultrapure water, uniformly mixing, and then placing into a shaking table for incubation reaction. Wherein the pH of the 2 XPBS is from 5.0 to 9.0, preferably any one of 5.0, 5.5, 6.0, 6.5, 7.4, 8.0, 8.5 or 9.0, more preferably the pH of the 2 XPBS is 5.0; the amount of Protein L added is 5-15% of the amount of pretreatment material, preferably 5%, 10% or 15%, more preferably 5%; the incubation reaction temperature is 25+/-5 ℃, the incubation reaction time is 6-24h, and the incubation reaction time is preferably any one of 6h, 12h or 24h.
And S3, dispersing the protein-coupled nano magnetic beads in a second buffer solution for preservation.
Specifically, the second buffer comprises PBST having a pH of 7.4, optionally comprising 1 XPBS and 0.5% Tween-20; the preservation temperature is 4+/-1 ℃ and the concentration of the magnetic beads is 10mg/mL. The protein-coupled nano magnetic beads are dispersed in PBST to prepare 10mg/mL protein-coupled nano magnetic beads, and the 10mg/mL protein-coupled nano magnetic beads are stored at 4+/-1 ℃, so that the final product (namely the protein-coupled nano magnetic beads) has long storage time and good biological activity.
Therefore, the polydopamine coated outside the magnetic ferric oxide particles contains a large number of functional groups such as amino, hydroxyl, carbonyl and the like, can be chelated and anchored on most inorganic and organic materials, can also be used as an intermediate medium for subsequent reaction of the materials, and provides rich organic functional groups for functional modification of the materials. Compared with the traditional chemical coupling method, the preparation method has the characteristics of mild reaction conditions, simple preparation steps, low production cost, short period and the like, and the prepared final product (namely the Protein coupled nano magnetic beads) has good biological activity, high purification efficiency, long storage time and recycling.
Further, in step S1, the polydopamine-coated magnetic iron oxide particles of the present embodiment are obtained by self-making, and the preparation of the polydopamine-coated magnetic iron oxide particles specifically includes the following steps:
s11, self-making magnetic iron oxide nano particles;
specifically, the magnetic iron oxide nano-particles are ferroferric oxide nano-materials and/or metal ion doped ferroferric oxide nano-materials, wherein the metal ions are any one of magnesium ions, cobalt ions, nickel ions or zinc ions, and the average particle size of the magnetic iron oxide nano-particles is about 200nm. Preferably, taking metal ion as magnesium ion as an example, mixing ferric chloride hexahydrate, magnesium chloride hexahydrate, anhydrous sodium acetate, ethylene glycol and polyethylene glycol 600, stirring uniformly, and then placing into a reaction kettle for high-temperature treatment to obtain Mg-Fe 3 O 4 Wherein the stirring time is 12 hours, the reaction temperature is 200 ℃, and the high-temperature reaction time is 12 hours.
S12, performing magnetic separation and washing treatment on the magnetic iron oxide nano particles, and then dispersing the magnetic iron oxide nano particles in a solvent by ultrasonic to obtain a pretreated material;
in particular, magnetic iron oxide nanoparticles (e.g., mg—fe 3 O 4 ) And magnetically separating the supernatant, washing the supernatant at least once with ultrapure water, and dispersing the supernatant in ethanol to obtain a pretreated material.
And S13, adding dopamine for polymerization to obtain polydopamine-coated magnetic iron oxide particles.
Specifically, quantitatively drying the pretreated material, then washing the quantitatively dried pretreated material at least once, magnetically separating the supernatant, and placing the supernatant into a centrifuge tube; adding ultrapure water, a second buffer solution and dopamine, mixing uniformly by ultrasonic, and putting into a shaking table for shaking reaction. Wherein the second buffer solution comprises Tris-HCl, and the pH value of the Tris-HCl is 8.5; the input amount of the dopamine is 5-75% of the dosage of the pretreatment materials, and the input amount of the dopamine is preferably any one of 5%, 10%, 15%, 25%, 35%, 50% or 70%, and more preferably the input amount of the dopamine is 25%; the shaking reaction temperature is 35.+ -. 5 ℃, the rotation speed is 200rpm, the polymerization reaction time is 6-24h, and the polymerization reaction time is preferably any one of 6h, 12h or 24h.
The embodiment also provides an application of the protein-coupled nanomagnetic beads, which has the preparation method of the protein-coupled nanomagnetic beads, and the protein-coupled nanomagnetic beads prepared by the preparation method can be used for purifying IgG in a target object, wherein the target object is any one of mixed solution of fetal Bovine Serum (BSA) and IgG, mixed solution of cell lysate and IgG or human serum. Therefore, the protein-coupled magnetic nanoparticle prepared in the embodiment has the adsorption capacity of human immunoglobulin G reaching about 10% (W/W), and has excellent selectivity and higher purification efficiency.
Example 1
In this embodiment, taking metal ion as magnesium ion as an example, a preparation method of protein-coupled nano magnetic beads includes the following steps:
(1) 270.0mg of ferric trichloride hexahydrate, 80mg of magnesium chloride hexahydrate, 1.0g of anhydrous sodium acetate, 10mL of polyethylene glycol and 10mL of polyethylene glycol 600 are mixed, and the materials are stirred uniformly by a magnetic stirrer for 12 hours; the mixture is put into a reaction kettle for high-temperature treatment, the reaction temperature is 200 ℃ and the reaction time is 12 hours, and the magnesium doped ferroferric oxide, namely Mg-Fe, is obtained 3 O 4 。
(2) Mg-Fe 3 O 4 Separating the supernatant by magnetism, washing the supernatant once with ultrapure water, and dispersing the washed supernatant in 3mL of ethanol to obtain a pretreated material; subsequently, the pretreated material is dried and quantified; then 5mg of the pretreated material after the drying treatment is taken out and washed once, the supernatant is separated magnetically and put into a centrifuge tube, 2.5mL of ultrapure water, 2.5mL of Tris-Cl (1M, pH is 8.5) and 1.25mg of dopamine (the input ratio is 25 percent) are added, and the mixture is mixed evenly by ultrasound and put into a shaking table for oscillating reaction (the reaction temperature is 35℃,Rotating speed of 200rpm and reaction time of 24 hours) to obtain Mg-Fe 3 O 4 @PDA。
(3) Mg-Fe 3 O 4 Washing the PDA once, and dispersing the PDA in 2mL PBS to obtain a pretreatment material; taking out 0.5mL of the pretreated material, placing the pretreated material into a centrifuge tube, magnetically separating the supernatant, and adding 0.5mL of 2 XPBS (pH 5.0), 0.5mg of Protein L (input ratio 10%) and 0.5mL of ultrapure water; then the mixture is inverted and evenly mixed and put into a shaking table for reaction, the reaction temperature is 25 ℃, the rotating speed is 200rpm, the reaction time is 12 hours, the supernatant is separated by magnetism after the reaction is finished, and the supernatant is dispersed in 0.5mL of PBST to prepare 10Mg/mL of Mg-Fe 3 O 4 @PDA-Protein L。
FIG. 1 shows the Mg-Fe prepared by this example 3 O 4 Nanometer magnetic bead and Mg-Fe 3 O 4 From the SEM image of PDA nano magnetic beads, it can be seen from fig. 1 that the nano magnetic beads have uniform size and good monodispersity, the average particle diameter is about 200nm, and the surfaces of the magnetic beads become more blurred after the polydopamine polymer is coated, mainly due to the non-conductivity of polydopamine. FIG. 2 shows the Mg-Fe prepared by example 1 3 O 4 From the TEM image of PDA nanomagnetic beads, a layer of amorphous material was clearly observed on the surface of the beads, which further confirmed that polydopamine macromolecules were successfully encapsulated on the surface of the nanomagnetic beads. FIG. 3 shows the Mg-Fe prepared by example 1 3 O 4 Nanometer magnetic bead and Mg-Fe 3 O 4 IR diagram of PDA nanomagnetic beads, from FIG. 3, it can be seen that Mg-Fe 3 O 4 The nanometer magnetic beads are 550cm in length -1 The wide absorption peak of Fe-O bond characteristic appears at the position, and after the poly-dopamine macromolecule is wrapped, the magnetic beads are respectively at 2930cm -1 、1602cm -1 1486cm -1 Three characteristic peaks are added, which are respectively the telescopic shock absorption peaks of C-H bond and aromatic ring C-C bond characteristics in the polydopamine macromolecule, which further proves that the polydopamine macromolecule is successfully wrapped on the surface of the nano magnetic bead.
Example 2
The present example differs from example 1 in that the dopamine investment ratio in step (2) is any one of 5%, 10%, 15%, 50% or 75%.
Examples 1 to 2 Mg-Fe 3 O 4 The results of purifying IgG by PDA-Protein L nano magnetic beads under different input amounts of dopamine are shown in Table 1.
TABLE 1 results of purification of IgG by nanomagnetic beads with different dopamine inputs
Note that: magnetic bead purification efficiency (%) = (first gray value-second gray value)/first gray value×100%, wherein the first gray value is the gray value of IgG in mixed solution protein gel electrophoresis of fetal Bovine Serum (BSA) and IgG, and the second gray value is the gray value of IgG in supernatant protein gel electrophoresis of nano magnetic beads after purifying IgG in mixed solution of fetal Bovine Serum (BSA) and IgG.
As can be seen from Table 1, the magnetic bead purification efficiency can reach 9.5% or more when the dopamine input ratio is 25% or 50%; especially, when the input ratio of the dopamine is 25%, the purification efficiency of the magnetic beads can reach 11.5%, and the higher purification efficiency is shown.
Example 3
This example differs from example 1 in that the reaction time for the polymerization of dopamine in step (2) is 6 hours or 24 hours.
Examples 1 and 3 prepared Mg-Fe 3 O 4 The results of purifying IgG with PDA-Protein L nanomagnetic beads at different polymerization (encapsulation) times of dopamine are shown in table 2.
TABLE 2 results of purification of IgG by nanomagnetic beads at different polymerization times of dopamine
As can be seen from Table 2, when the polymerization (encapsulation) time of dopamine is any one of 6h, 12h or 24h, the purification efficiency of the magnetic beads can reach more than 10%, and the higher purification efficiency is shown.
Example 4
This example differs from example 1 in that Mg-Fe 3 O 4 The conditions for the @ PDA conjugate protein are different, i.e. the pH of the 2 x PBS in step (3) is different, in this example the pH of the 2 x PBS may be any of 5.5, 6.0, 6.5, 7.4, 8.0, 8.5 or 9.0.
Examples 1 and 4 prepared Mg-Fe 3 O 4 The results of purifying IgG with PDA-Protein L nanomagnetic beads under different coupling conditions are shown in table 3.
TABLE 3 results of purification of IgG from nanomagnetic beads under different coupling conditions
Note that: igG purification efficiency (%) = (total amount of adsorbed IgG/total amount of IgG input) ×100%; magnetic bead adsorption IgG efficiency (%) = (IgG adsorption total amount/nano magnetic bead input total amount) ×100%.
As can be seen from Table 3, under the condition that the total input amount of 100 mug of nano magnetic beads and the total input amount of 20 mug of IgG are 5.0, 5.5, 6.0, 6.5, 7.4 or 8.0 of the coupling Protein L reaction solution pH, the adsorption efficiency of the magnetic beads to the IgG can reach more than 9.2 percent; in particular, the magnetic beads prepared under the condition of pH 5.0 have the IgG adsorption efficiency reaching 9.92 percent (nearly 10 percent), and show higher purification efficiency.
Example 5
This example differs from example 1 in that Mg-Fe 3 O 4 The conditions for the @ PDA conjugated Protein are different, i.e., the amount of Protein L charged in step (3) is different, and in this example, the charged Protein L may be 10% or 15%.
Examples 1 and 5 prepared Mg-Fe 3 O 4 The results of purifying IgG at different amounts of Protein L by PDA-Protein L nanomagnetic beads are shown in Table 4.
TABLE 4 purification of IgG by nanomagnetic beads at different inputs of Protein L
As can be seen from Table 4, when the input ratio of Protein L was 5%, the magnetic bead purification efficiency was 10% or more, and higher purification efficiency was exhibited.
Example 6
This example differs from example 1 in that Mg-Fe 3 O 4 The conditions for coupling the Protein at PDA are different, i.e.the Protein L coupling time in step (3) is different, in this example the Protein L coupling time (i.e.reaction time) may be 6h or 24h.
Examples 1 and 6 prepared Mg-Fe 3 O 4 The results of purifying IgG with PDA-Protein L nanomagnetic beads at different coupling times of Protein L are shown in table 5.
TABLE 5 results of purification of IgG by nanomagnetic beads at different coupling times of Protein L
As can be seen from Table 5, when the polymerization (encapsulation) time of dopamine is either 12 hours or 24 hours, the purification efficiency of the magnetic beads can reach more than 10%, and the purification efficiency is high.
Example 7
To investigate the purified IgG properties of the prepared nanomagnetic bead material, mg-Fe prepared in any one of examples 1 to 6 3 O 4 The purification of IgG Protein was performed on PDA-Protein L nanomagnetic beads. To prepare the Mg-Fe prepared in example 1 3 O 4 For example, PDA-Protein L nanomagnetic beads were subjected to an IgG Protein purification experiment, and before the IgG Protein purification experiment was performed, the nanomagnetic bead material prepared in example 1 was subjected to purification classification, which includes, but is not limited to, any of the following:
(1) Mg-Fe prepared in example 1 3 O 4 Purifying IgG in a mixed solution of BSA and IgG by using the @ PDA-Protein L nano magnetic beads;
(2) Mg-Fe prepared in example 1 3 O 4 Purifying IgG in a mixed solution of bacterial lysate and IgG by using the PDA-Protein L nano magnetic beads;
(3) Mg-Fe prepared in example 1 3 O 4 The @ PDA-Protein L nano magnetic beads purified IgG in human serum.
1. By adopting the classification conditions, the prepared nano magnetic bead material is used for carrying out an IgG protein purification experiment under the classification condition (1), and the reaction conditions are as follows:
1.1 taking 300. Mu.g of Mg-Fe prepared in example 1 3 O 4 Mixing @ PDA-Protein L with 10 μg IgG, shaking, mixing for 2 hr, and collecting supernatant for temporary storage. The nano magnetic beads are washed once by 15 mu L of PBST cleaning liquid and then temporarily stored as the cleaning liquid supernatant. The nano magnetic beads are washed once by 15 mu L of Gly eluent to be used as the supernatant of the primary eluent, and finally the nano magnetic beads are washed once by 15 mu L of Gly eluent to be used as the supernatant of the secondary eluent. Wherein the PBST comprises 1 XPBS and 1% Tween-20, and the pH value of the PBST is 7.4; gly is 0.3M Glycine and its pH is 2.5.
1.2A mixed solution of 5mg of IgG and 5mg of BSA in 500. Mu.L of PBST was used as a reference sample, and the supernatant obtained in step 1.1 was used as a measurement set and added to SDS-PAGE gel to carry out protein gel electrophoresis. The isolation gel was run at 80V for 30min, followed by concentration gel run at 120V for 2h. After the electrophoresis, the sample was stained with Coomassie brilliant blue (staining time: 30min, staining temperature: 37 ℃ C., rotation speed: 80 rpm), and after the staining was completed, the sample was subjected to decolorization and observation.
FIG. 4 shows the Mg-Fe mixture obtained in example 1 3 O 4 SDS-page diagram of purifying IgG by PDA-Protein L nano magnetic beads in a mixture of BSA and IgG, wherein in the diagram, line 1 is Marker, line 2 is mixed solution of BSA and IgG, line 3 is supernatant of the nano magnetic beads after purifying the mixed solution of BSA and IgG, line 4 is supernatant of the magnetic beads after washing by PBST washing liquid, and Line 5 is supernatant of the magnetic beads after eluting by Gly eluent. As can be seen from FIG. 4, the Gly eluent can elute part of IgG, and the prepared Mg-Fe 3 O 4 The @ PDA-Protein L nano magnetic beads can effectively purify IgG in a mixture of BSA and IgG.
2. By adopting the classification conditions, the prepared nano magnetic bead material is used for carrying out an IgG protein purification experiment under the classification condition (2), and the reaction conditions are as follows:
2.1 sucking 100 mu L of coliform bacteria liquid, adding the liquid into a culture medium, and incubating for 12 hours at 37 ℃ and 100 rpm; centrifuging to remove supernatant (rotating speed 3000rpm, time 5 min) after the reaction is finished to obtain bacterial liquid; the obtained bacterial liquid is centrifugally washed by using a large amount of ultrapure water, and the bacterial liquid is centrifugally washed for 2 times under the condition that the rotating speed is 3000rpm, and each time is 3 minutes; then dispersing the cells in PBST, and using an ultrasonic breaker to lyse the cells, wherein the ultrasonic treatment time is 2-4min, and the ultrasonic frequency is 10kHZ; centrifuging to obtain supernatant (rotation speed 10000rpm, time 10 min); 1.5:1, mixing the supernatant with IgG to obtain a mixed solution.
2.2 taking 300. Mu.g of Mg-Fe prepared in example 1 3 O 4 PDA-Protein L is washed once by 600 mu L of PBST cleaning solution, supernatant is magnetically separated, and is mixed with 10 mu g of IgG, and is vibrated and mixed for 2 hours, and supernatant after reaction is temporarily stored. The nano magnetic beads are washed once by 15 mu L of PBST cleaning liquid and then temporarily stored as the cleaning liquid supernatant. The nano magnetic beads are washed once with 15 mu L of Gly eluent to be used as the supernatant of the primary eluent, and finally the nano magnetic beads are washed once with 15 mu L of Gly eluent to be used as the supernatant of the secondary eluent.
2.3 taking 10 mu L of the mixed solution obtained in the step 2.1 as a reference sample, adding the supernatant obtained in the step 2.2 as a measurement group into an SDS-PAGE gel hole, carrying out protein gel electrophoresis, running the separation gel at 80V for 30min, and then running the concentrated gel at 120V for 2h; after the electrophoresis, the sample was stained with Coomassie brilliant blue (staining time: 30min, staining temperature: 37 ℃ C., rotation speed: 80 rpm), and after the staining was completed, the sample was subjected to decolorization and observation.
FIG. 5 shows the Mg-Fe mixture obtained in example 1 3 O 4 SDS-page diagram of purifying IgG by PDA-Protein L nanometer magnetic beads in a mixture of cell lysate and IgG, wherein in the diagram, line 1 is Marker, line 2 is cell lysate, line 3 is mixture of cell lysate and IgG, line 4 is supernatant of purifying nanometer magnetic beads in the mixture of cell lysate and IgG, line 5 is supernatant of cleaning magnetic beads by PBST cleaning solution, line 6 is supernatant of eluting magnetic beads by Gly eluent, and Line 7 is Gly elutionThe supernatant after the beads were eluted again with Line 8 as Marker. As can be seen from FIG. 5, the Gly eluent can elute part of IgG, and the prepared Mg-Fe 3 O 4 The @ PDA-Protein L nano magnetic beads can effectively purify IgG in a mixture of cell lysate and IgG.
3. By adopting the classification conditions, the prepared nano magnetic bead material is used for carrying out an IgG protein purification experiment under the classification condition (3), and the reaction conditions are as follows:
3.1, using a vacuum tube containing separation gel, taking whole blood by an elbow vein blood sampling method, standing for 30min, and centrifuging to obtain supernatant (with the rotating speed of 3000rpm and the time of 5 min); the supernatant protein was quantified and the serum IgG concentration interval was calculated. 300. Mu.g of Mg-Fe prepared in example 1 was taken 3 O 4 PDA-Protein L is washed once by 600 mu L of PBST cleaning solution, supernatant is magnetically separated, and is mixed with 10 mu g of IgG, and is vibrated and mixed for 2 hours, and supernatant after reaction is temporarily stored. The nano magnetic beads are washed once by 15 mu L of PBST cleaning liquid and then temporarily stored as the cleaning liquid supernatant. The nano magnetic beads are washed once with 15 mu L of Gly eluent to be used as the supernatant of the primary eluent, and finally the nano magnetic beads are washed once with 15 mu L of Gly eluent to be used as the supernatant of the secondary eluent.
3.2 taking 10 mu L of the mixed solution obtained in the step 3.1 as a reference sample, adding the supernatant obtained in the step 3.1 as a measurement group into SDS-page gel holes, carrying out protein gel electrophoresis, running the separation gel at 80V for 30min, and then running the concentrated gel at 120V for 2h. After the electrophoresis, the sample was stained with Coomassie brilliant blue (staining time: 30min, staining temperature: 37 ℃ C., rotation speed: 80 rpm), and after the staining was completed, the sample was subjected to decolorization and observation.
FIG. 6 shows the Mg-Fe mixture obtained in example 1 3 O 4 SDS-page diagram of purifying IgG by PDA-Protein L nanometer magnetic beads in human serum, wherein in the diagram, line 1 is Marker, line 2 is IgG, line 3 is human serum, line 4 is supernatant of purifying nanometer magnetic beads in human serum, line 5 is supernatant of PBST cleaning solution magnetic beads, line 6 is supernatant of eluting magnetic beads by Gly eluent, and Line 7 is Marker. As can be seen from FIG. 6, gly eluent can elute part of IgG, and Mg-Fe is prepared 3 O 4 The @ PDA-Protein L nano magnetic beads can effectively purify IgG in human serum.
Therefore, the prepared protein-coupled nano magnetic beads have higher purification efficiency and excellent selectivity on human immunoglobulin G, and can specifically separate and purify the human immunoglobulin G in fetal bovine serum, cell lysate or human serum.
What has been described above is merely some embodiments of the present invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.
Claims (10)
1. The preparation method of the protein-coupled nano magnetic beads is characterized by comprising the following steps:
s1, washing polydopamine coated magnetic iron oxide particles, and dispersing the magnetic iron oxide particles in a first buffer solution to obtain a pretreatment material;
s2, adding target protein for incubation treatment, and magnetically separating supernatant after incubation reaction to obtain protein-coupled nano magnetic beads;
and S3, dispersing the protein-coupled nano magnetic beads in a second buffer solution for preservation.
2. The method of claim 1, wherein the first buffer solution comprises PBS; the specific steps of dispersing the polydopamine-coated magnetic iron oxide particles in the first buffer solution after washing comprise:
after washing the polydopamine coated magnetic iron oxide particles at least once with ultrapure water, the polydopamine coated magnetic iron oxide particles are ultrasonically dispersed in PBS.
3. The method for preparing protein-coupled nanomagnetic beads according to claim 1, wherein said added target protein comprises proteonl; the specific steps of adding target protein for incubation treatment comprise:
taking a quantitative pretreatment material, magnetically separating supernatant, adding 2 XPBS, proteinL and ultrapure water, uniformly mixing, and then placing into a shaking table for incubation reaction.
4. The method for preparing protein-coupled nanomagnetic beads according to claim 3, wherein the pH value of the 2 XPBS is 5.0-9.0, the input amount of the ProteinL is 5-15% of the amount of the pretreatment material, the reaction temperature is 25+ -5 ℃, and the incubation reaction time is 6-24h.
5. The method of claim 1, wherein the second buffer comprises PBST, the PBST comprising 1 x PBS and 0.5% tween-20; the preservation temperature is 4+/-1 ℃ and the concentration of the magnetic beads is 10mg/mL.
6. The method for preparing the protein-coupled nanomagnetic beads according to claim 1, wherein the preparation of the polydopamine-coated magnetic iron oxide particles specifically comprises the following steps:
s11, self-making magnetic iron oxide nano particles;
s12, performing magnetic separation and washing treatment on the magnetic iron oxide nano particles, and then dispersing the magnetic iron oxide nano particles in a solvent by ultrasonic to obtain a pretreated material;
and S13, adding dopamine for polymerization to obtain polydopamine-coated magnetic iron oxide particles.
7. The method for preparing protein-coupled nanomagnetic beads according to claim 6, wherein the magnetic iron oxide nanoparticles are ferroferric oxide nanomaterials and/or metal ion doped ferroferric oxide nanomaterials, wherein the metal ions are any one of magnesium ions, cobalt ions, nickel ions or zinc ions; the input amount of the dopamine is 5-75% of the amount of the pretreatment material, and the polymerization reaction time is 6-24 hours.
8. The method for preparing protein-coupled nanomagnetic beads according to claim 6 or 7, wherein the specific steps of self-made magnetic iron oxide nanoparticles include:
mixing ferric chloride hexahydrate, magnesium chloride hexahydrate, anhydrous sodium acetate, ethylene glycol and polyethylene glycol 600, stirring uniformly, and then placing into a reaction kettle for high-temperature treatment.
9. The method for preparing protein-coupled nanomagnetic beads according to claim 6 or 7, wherein the specific step of adding dopamine for polymerization comprises:
washing a quantitative pretreatment material, magnetically separating a supernatant, adding a second buffer solution and dopamine, uniformly mixing by ultrasound, and then putting into a shaking table for oscillating reaction.
10. Use of protein-coupled nanomagnetic beads according to any one of claims 1-9 for purification of IgG in a target, wherein the target is any one of fetal Bovine Serum (BSA) and IgG mixed solution, cell lysate and IgG mixed solution, or human serum.
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