CN118022690B - Surface-modified magnetic bead and preparation method and application thereof - Google Patents
Surface-modified magnetic bead and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a surface-modified magnetic bead, a preparation method and application thereof; belongs to the technical field of immunity; the preparation method comprises the following steps: providing nano-scale Fe 3O4 magnetic beads; the nanoscale Fe 3O4 magnetic beads react with a silane coupling agent to carry an amino group to obtain amino-modified magnetic beads; reacting the amino-modified magnetic beads with an aldehyde compound to obtain surface-modified magnetic beads; coupling the surface modified magnetic beads with a chitinase 3-like protein 1 (CHI 3L 1) polyclonal antibody to obtain chitinase 3-like protein 1 (CHI 3L 1) immunomagnetic beads; wherein the aldehyde compound comprises 2-hexenal. The surface modified magnetic beads prepared by the method have low protein nonspecific adsorption capacity, and the immune magnetic beads have high detection sensitivity on chitinase 3-like protein 1 (CHI 3L 1).
Description
Technical Field
The invention relates to the technical field of immunity, in particular to a surface modified magnetic bead, a preparation method and application thereof.
Background
In the eighth nineties of the twentieth century, foreign researchers adopted different monomers to synthesize polymer microspheres with different surface morphologies, wherein the macroporous microspheres absorb magnetic particles through activation and swelling, the magnetic particles are uniformly distributed in the whole microsphere in the form of small particles, and finally commercial Dynabeads series magnetic beads are synthesized, and the series magnetic beads are well applied in the aspects of cell separation, nucleic acid extraction, immunoassay and the like. The company producing the immunomagnetic beads with high commercialization degree starts from Dynabeads of Norway, but also has Bangs beads in the United states, JSR beads in Japan, FG beads and the like, and the domestic use of the immunomagnetic beads is seriously dependent on import, and although enterprises and universities are researching the preparation of the immunomagnetic beads, the product performance is not high, the difference between production batches is large, so that the research of the preparation method is necessary to grasp core technology, and finally the immunomagnetic beads with excellent preparation performance replace foreign products.
The magnetic sources of the magnetic nano particles can be metals such as iron, cobalt, nickel and the like, but pure metals cannot be stably oxidized in the air, so that the subsequent surface modification work can be influenced, wherein the ferroferric oxide (Fe 3O4) nano particles obtained after the oxidation of the elemental iron gradually become hot spots for research of the magnetic nano materials at home and abroad due to the characteristics of high comparison area, low toxicity, good magnetic response, biocompatibility and the like. However, the high specific surface area tends to make the particles have high surface energy, so that the Fe 3O4 nano particles are unstable and easy to agglomerate, the monodispersity and stability of the nano particles can be improved through surface modification, and the magnetic particles after surface modification can be further modified through a chemical method according to the requirement and can be applied to various fields.
Disclosure of Invention
The invention aims to provide an aldehyde compound which is reacted with magnetic beads with amino groups, so that the prepared surface modified magnetic beads have low nonspecific adsorption capacity to proteins, and the detection sensitivity to chitinase 3-like protein 1 (CHI 3L 1) is high after the prepared double-antibody sandwich immunomagnetic beads are prepared.
The technical scheme adopted by the invention for achieving the purpose is as follows:
A method for preparing surface-modified magnetic beads comprises providing nanoscale Fe 3O4 magnetic beads; the nano Fe 3O4 magnetic beads react with a silane coupling agent to carry an amino group; adding amino-modified Fe 3O4 magnetic beads into aldehyde compound solution for reaction, performing magnetic separation, and washing to obtain the surface-modified magnetic beads.
Preferably, the silane coupling agent comprises 3-aminopropyl trimethoxysilane.
Preferably, the aldehyde compound solution comprises a 2-hexenal solution; the Fe 3O4 magnetic bead with modified amino reacts with 2-hexenal, and when the prepared surface modified magnetic bead is used in the biological field, the surface of the surface modified magnetic bead can well inhibit the nonspecific adsorption of protein.
Preferably, the nano-scale Fe 3O4 magnetic beads are 20-60 parts by weight.
Preferably, the volume part of the silane coupling agent is 100 to 300 parts.
Preferably, the mass fraction of the 2-hexenal solution is 20-30%.
Preferably, the weight part of the amino-modified Fe 3O4 magnetic beads is 2-10 parts.
Preferably, the aldehyde compound solution is 1000-2000 parts by volume.
Preferably, the condition of the reaction of the nano-scale Fe 3O4 magnetic beads and the silane coupling agent is that the stirring speed is 200-400 r/min, and the reaction is 8-12 h at the temperature of 0-70 ℃.
Preferably, the condition of the reaction of the amino-modified Fe 3O4 magnetic beads added into the aldehyde compound solution is that the reaction is carried out at room temperature of 4-8 h.
The invention also provides a surface-modified magnetic bead, which is prepared by the preparation method of the surface-modified magnetic bead.
The invention also provides an immune magnetic bead, which comprises an immune ligand and the surface modified magnetic bead prepared by any one of the preparation methods or the surface modified magnetic bead, wherein the immune ligand is combined with the surface modified magnetic bead, and the immune ligand comprises a chitinase 3-like protein 1 (CHI 3L 1) polyclonal antibody.
The invention also provides a kit for liver disease detection, and the reagent in the kit for liver disease detection contains the surface modified magnetic beads or the immunomagnetic beads.
In one embodiment, a surface-modified magnetic bead and a method for preparing the same comprises the steps of:
firstly, preparing nano-scale Fe 3O4 magnetic beads, putting FeCl 3·6H2 O and FeCl 2·4H2 O into a conical flask, adding ultrapure water, stirring uniformly at room temperature, adding NaOH, stirring 20-40 min, heating to 60-70 ℃, and aging 25-35 min; after the reaction is finished, performing magnetic separation under the action of an external magnetic field, and washing with ultrapure water for several times until the mixture is neutral to obtain nano-scale Fe 3O4 magnetic beads; wherein, the weight part of FeCl 3·6H2 O is 300-500 parts, and the weight part of FeCl 2·4H2 O is 200-300 parts; 250-350 parts of NaOH; the volume of the ultrapure water is 10000-30000 parts by volume.
Further, the dosage ratio of FeCl 3·6H2O、FeCl2·4H2 O to ultrapure water is 3-5 g:2-3 g:100-200 mL.
Preparing amino-modified Fe 3O4 magnetic beads, adding the nano-scale Fe 3O4 magnetic beads synthesized in the step one into absolute ethyl alcohol, adding 3-aminopropyl trimethoxysilane, and reacting at 50-70 ℃ for 8-12 h under the condition of stirring speed of 250-350 r/min and nitrogen inlet. After the reaction is finished, performing magnetic separation under the action of an external magnetic field, washing 3-5 times by using absolute ethyl alcohol, and then washing 3-5 times by using ultrapure water to obtain Fe 3O4 magnetic beads with modified amino groups; wherein, the weight part of the nano-scale Fe 3O4 magnetic beads is 20-60 parts; the volume of the absolute ethyl alcohol is 4000-6000 parts by volume; the volume part of the 3-aminopropyl trimethoxy silane is 100-300 parts.
Further, the dosage ratio of the nano-scale Fe 3O4 magnetic beads to the 3-aminopropyl trimethoxysilane is 0.2-0.6 g:40-50 mL.
Preparing surface modified magnetic beads, dispersing the amino-modified Fe 3O4 magnetic beads prepared in the second step by using PBS, carrying out ultrasonic treatment for 20-40min to obtain amino-modified Fe 3O4 magnetic bead solution, adding 2-hexenal solution, stirring at room temperature for 4-8 h under the condition of introducing nitrogen, carrying out magnetic separation under the action of an external magnetic field after the reaction is finished, and washing for 3-5 times to obtain the surface modified magnetic beads; wherein, the weight part of the Fe 3O4 magnetic beads for modifying amino is 2-10 parts; the concentration of PBS is 0.01-0.02M, pH=7-8, and the volume of PBS is 300-500; the mass fraction of the 2-hexenal solution is 20-30%, and the volume fraction is 1000-2000%.
Further, the dosage ratio of the Fe 3O4 magnetic beads for modifying amino to the 2-hexenal solution is 20-100 mg:10-20 mL.
Preferably, preparing surface modified magnetic beads, dispersing the amino-modified Fe 3O4 magnetic beads prepared in the step two by PBS, carrying out ultrasonic treatment for 20-40min to obtain amino-modified Fe 3O4 magnetic bead solution, adding hydroxy-1, 4-succinyl aldehyde solution, stirring at room temperature for 4-8 h under the condition of introducing nitrogen, carrying out magnetic separation under the action of an external magnetic field after the reaction is finished, and washing for 3-5 times to obtain the surface modified magnetic beads; wherein, the weight part of the Fe 3O4 magnetic beads for modifying amino is 2-10 parts; the concentration of PBS is 0.01-0.02M, pH=7-8, and the volume of PBS is 300-500; the mass fraction of the hydroxy-1, 4-butanedial solution is 20-30%, and the volume part is 1000-2000; hydroxyl-1, 4-butanedialdehyde is used for replacing 2-hexenal to react with Fe 3O4 magnetic beads for modifying amino to obtain surface modified magnetic beads, so that nonspecific adsorption of proteins can be further reduced; after the chitosan 3-like protein is prepared into the double-antibody sandwich immunomagnetic beads, the combination of specific proteins (such as CHI3L 1) can be enhanced, and the detection sensitivity of the chitosan 3-like protein 1 (CHI 3L 1) is improved.
Further, the dosage ratio of the Fe 3O4 magnetic beads for modifying amino to the hydroxy-1, 4-butanedial solution is 20-100 mg/10-20 mL.
More preferably, preparing surface modified magnetic beads, dispersing the amino-modified Fe 3O4 magnetic beads prepared in the step two by PBS, performing ultrasonic treatment for 20-40min to obtain amino-modified Fe 3O4 magnetic bead solution, adding hydroxy-1, 4-succinyl aldehyde solution and 2-hexenal solution, stirring at room temperature under the condition of introducing nitrogen, stirring for 4-8 h at room temperature, performing magnetic separation under the action of an external magnetic field after the reaction is finished, and washing for 3-5 times to obtain the surface modified magnetic beads; wherein, the weight part of the Fe 3O4 magnetic beads for modifying amino is 2-10 parts; the concentration of PBS is 0.01-0.02M, pH=7-8, and the volume of PBS is 300-500; the mass fraction of the hydroxy-1, 4-butanedial solution is 20-30%, and the volume part of the hydroxy-1, 4-butanedial solution is 1000-2000; the mass fraction of the 2-hexenal solution is 20-30%, and the volume part of the 2-hexenal solution is 1000-2000; because the hydroxy-1, 4-butanedial and 2-hexenal are used simultaneously, the two aldehyde compounds can generate a synergistic effect, so that the nonspecific adsorption quantity of protein on the surface of the prepared surface-modified magnetic bead is reduced when the surface-modified magnetic bead is applied to the biological field; and after the CHI3L1 antibody is prepared into the double-antibody sandwich immunomagnetic beads, the detection sensitivity of the CHI3L1 protein is improved.
Further, the dosage ratio of the Fe 3O4 magnetic beads for modifying amino, the hydroxy-1, 4-butanedial solution and the 2-hexenal solution is 20-100 mg:10-20 mL:10-20 mL.
The invention also provides a kit (acridinium ester magnetic particle chemiluminescence method) for identifying and/or prognosis monitoring liver fibrosis and liver cirrhosis, which comprises the following reagents:
reagent 1: contains anti-CHI 3L1 monoclonal antibody with concentration of 0.2-1.0 mug/mL, which is treated by cross-linking agent, biotin or fluorescein; the monoclonal antibody comprises at least one of a mouse anti-human CHI3L1 monoclonal antibody, a sheep anti-human CHI3L1 monoclonal antibody and a rabbit anti-human CHI3L1 monoclonal antibody;
Reagent 2: comprises anti-CHI 3L1 polyclonal antibody marked by horseradish peroxidase, alkaline phosphatase or acridinium ester with the concentration of 0.2-1.0 mug/mL; the polyclonal antibody comprises at least one of a mouse anti-human CHI3L1 polyclonal antibody, a sheep anti-human CHI3L1 polyclonal antibody and a rabbit anti-human CHI3L1 polyclonal antibody;
reagent 3: the surface modified magnetic beads prepared above contain 0.5-2 mg/mL;
calibration material: contains recombinant CHI3L1 antigen;
quality control product 1 and quality control product 2: contains a non-recombinant chitinase 3-like protein 1 (CHI 3L 1) antigen;
fully dissolving the calibrator and the quality control product according to 0.25-1.0 mL per branch, and standing at room temperature for 15-30 min;
Sample dilution: protein buffer, proclin300;
Coupling buffer: PBST and/or TBST buffer.
According to the invention, the prepared nano Fe 3O4 magnetic beads are subjected to surface amination modification by using a silane coupling agent, and the prepared amino-modified Fe 3O4 magnetic beads are reacted with aldehyde compounds comprising hydroxy-1, 4-succinyl aldehyde and 2-hexenal to obtain the surface modification magnetic beads, so that the functional modification of the surfaces of the nano magnetic beads is realized, the prepared surface modification magnetic beads have low nonspecific adsorption capacity on proteins, and after the double-antibody sandwich immunomagnetic beads are prepared, the detection sensitivity on chitinase 3-like protein 1 (CHI 3L 1) is high, so that the nano magnetic beads have wider application potential in the fields of biological sample treatment, biological analysis, medical diagnosis and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
FIG. 1 is a surface-modified magnetic bead electron microscope image.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The Phosphate Buffer (PBS) composition in the following embodiments is: naCl 8.0 g,KCl 0.2 g,Na 2HPO4·12H2O 2.9 g,KH2PO4 0.2.2. 0.2 g, distilled water was added to 1000. 1000 mL.
Example 1: surface-modified magnetic bead and preparation method and application thereof
S1, preparing nano-scale Fe 3O4 magnetic beads, putting 4.78 g FeCl 3·6H2 O and 2.78 g FeCl 2·4H2 O into a conical flask, adding 200 mL ultrapure water, stirring uniformly at room temperature, adding 3.2 g NaOH, stirring 30 min, heating to 65 ℃, aging 30 min, performing magnetic separation under the action of an external magnetic field after the reaction is finished, and washing with the ultrapure water for several times to neutrality to obtain nano-scale Fe 3O4 magnetic beads;
S2, preparing amino-modified Fe 3O4 magnetic beads, adding 0.43-g-nanometer Fe 3O4 magnetic beads into 50 mL absolute ethyl alcohol, adding 2-mL-3-aminopropyl trimethoxysilane, and reacting at 60 ℃ for 10 h under the condition of introducing nitrogen at the stirring speed of 300 r/min. After the reaction is finished, performing magnetic separation under the action of an external magnetic field, washing for 4 times by using absolute ethyl alcohol, and then washing for 4 times by using ultrapure water to obtain amino-modified Fe 3O4 magnetic beads;
S3, preparing surface modified magnetic beads, dispersing 50 mg modified amino Fe 3O4 magnetic beads by using 4 mL PBS (pH=7.4), performing ultrasonic treatment on the mixture to obtain an amino modified Fe 3O4 magnetic bead solution after 30 min, adding 10 mL mass percent of 25% 2-hexenal solution, stirring at room temperature for 6 h under the condition of introducing nitrogen, performing magnetic separation under the action of an external magnetic field after the reaction is finished, and washing with ultrapure water for 4 times to obtain the surface modified magnetic beads.
Example 2: surface-modified magnetic bead and preparation method and application thereof
S1, preparing nanoscale Fe 3O4 magnetic beads, and performing the same as in example 1;
s2, preparing amino-modified Fe 3O4 magnetic beads, and performing the same as in example 1;
s3, preparing surface-modified magnetic beads, wherein the addition amount of the 2-hexenal solution with the mass fraction of 25% is adjusted to 20 mL, unlike in example 1.
Example 3: surface-modified magnetic bead and preparation method and application thereof
S1, preparing nanoscale Fe 3O4 magnetic beads, and performing the same as in example 1;
s2, preparing amino-modified Fe 3O4 magnetic beads, and performing the same as in example 1;
S3, preparing surface modified magnetic beads, which are different from the embodiment 1 and the embodiment 2, wherein 50mg modified amino Fe 3O4 magnetic beads are dispersed by using 4 mL PBS (pH=7.4), an amino modified Fe 3O4 magnetic bead solution is obtained after 30 min is subjected to ultrasonic treatment, 10mL mass percent of a 25% hydroxyl-1, 4-succinyl aldehyde solution is added, stirring is carried out at room temperature under the condition of introducing nitrogen, 6 h is carried out at room temperature, magnetic separation is carried out under the action of an external magnetic field after the reaction is finished, and the surface modified magnetic beads are obtained after 4 times of washing by using ultrapure water.
Example 4: surface-modified magnetic bead and preparation method and application thereof
S1, preparing nanoscale Fe 3O4 magnetic beads, and performing the same as in example 1;
s2, preparing amino-modified Fe 3O4 magnetic beads, and performing the same as in example 1;
S3, preparing surface-modified magnetic beads, wherein the addition amount of the hydroxy-1, 4-butanedial solution with the mass fraction of 25% is adjusted to 20 mL, unlike example 3.
Example 5: surface-modified magnetic bead and preparation method and application thereof
S1, preparing nanoscale Fe 3O4 magnetic beads, and performing the same as in example 1;
s2, preparing amino-modified Fe 3O4 magnetic beads, and performing the same as in example 1;
S3, preparing surface modified magnetic beads, wherein the difference from the embodiment 1-4 is that 50 mg modified amino Fe 3O4 magnetic beads are dispersed by 4 mL PBS (pH=7.4), the amino modified Fe 3O4 magnetic bead solution is obtained after 30 min is ultrasonically treated, 10 mL of hydroxyl-1, 4-succinyl aldehyde solution with the mass fraction of 25% and 10 mL of 2-hexenal solution with the mass fraction of 25% are added, stirring is carried out at room temperature under the condition of introducing nitrogen, 6h is carried out, after the reaction is finished, magnetic separation is carried out under the action of an external magnetic field, and the surface modified magnetic beads are obtained after 4 times of washing with ultrapure water.
Example 6: surface-modified magnetic bead and preparation method and application thereof
S1, preparing nanoscale Fe 3O4 magnetic beads, and performing the same as in example 1;
s2, preparing amino-modified Fe 3O4 magnetic beads, and performing the same as in example 1;
S3, preparing surface-modified magnetic beads, wherein the addition amount of a hydroxyl-1, 4-butanedial solution with the mass fraction of 25% is adjusted to be 20 mL, unlike in example 5; the addition amount of the 2-hexenal solution with the mass fraction of 25% is adjusted to be 20 mL.
Example 7: kit for identifying and/or prognosis monitoring hepatic fibrosis and cirrhosis
The kit for identifying and/or prognosis monitoring liver fibrosis and cirrhosis (acridinium ester magnetic particle chemiluminescence method) comprises the following reagents:
Reagent 1: contains anti-CHI 3L1 monoclonal antibody with concentration of 0.5 mug/mL, which is treated by cross-linking agent and biotin; the monoclonal antibody comprises a mouse anti-human CHI3L1 monoclonal antibody;
Reagent 2: contains anti-CHI 3L1 polyclonal antibody marked by horseradish peroxidase with the concentration of 0.5 mug/mL; the polyclonal antibodies include murine anti-human CHI3L1 polyclonal antibodies;
Reagent 3: the surface-modified magnetic beads prepared in example 6 contained a concentration of 1 mg/mL;
calibration material: contains recombinant CHI3L1 antigen;
quality control product 1 and quality control product 2: contains recombinant chitinase 3-like protein 1 (CHI 3L 1) antigen;
Fully dissolving the calibrator and the quality control product according to 0.50 mL per branch, and standing at room temperature for 20: 20 min;
Sample dilution: protein buffer, proclin300;
coupling buffer: PBST and TBST buffers.
Comparative example 1:
S1, preparing nano-scale Fe 3O4 magnetic beads, putting 4.78 g FeCl 3·6H2 O and 2.78 g FeCl 2·4H2 O into a conical flask, adding 200 mL ultrapure water, stirring uniformly at room temperature, adding 3.2 g NaOH, stirring 30 min, heating to 65 ℃, aging 30 min, performing magnetic separation under the action of an external magnetic field after the reaction is finished, and washing with the ultrapure water for several times to neutrality to obtain nano-scale Fe 3O4 magnetic beads.
Test example 1:
1. transmission electron microscope characterization
1.1 Methods and samples
The magnetic polymer composite microspheres prepared in example 6 were observed with a Transmission Electron Microscope (TEM). The sample treatment steps were as follows: dispersing a proper amount of magnetic polymer composite microspheres in a centrifuge tube containing ethanol, uniformly dispersing the microspheres in the ethanol by using ultrasound, then dripping microsphere solution on a duplex copper mesh by using a pipetting gun, naturally airing, folding the copper mesh in half, and then observing by using a scanning electron microscope. Because the magnetic microsphere has superparamagnetism, a duplex copper mesh is needed, so that the sample is arranged between two layers of copper meshes, and the transmission electron microscope is prevented from being damaged.
Fig. 1 is a TEM image of a surface-modified magnetic bead prepared in example 6, in which the presence of a surface-modified layer of the magnetic bead is observed, the surface-modified layer is a uniform and dense layer of material covering the surface of the core of the magnetic bead, and the particles of the magnetic bead are densely and uniformly arranged and dispersed, so that it can be inferred that the magnetic bead has good dispersibility, which indicates that the embodiment of the present invention successfully performs surface modification on the nano-scale Fe 3O4 magnetic bead.
2. Comparison of nonspecific adsorption of magnetic beads on serum alpha-fetoprotein before and after surface modification by Coomassie Brilliant blue method
2.1, Samples and methods
(1) Preparing a coomassie brilliant blue reagent, dissolving 100 mg coomassie brilliant blue G-250 in a beaker, adding 50% mL% ethanol by mass for dissolution, continuously adding 85% phosphoric acid by mass of 100 mL%, transferring to a 1000 mL volumetric flask, and diluting to a scale with distilled water to obtain a 1000 mL solution;
(2) Preparing a standard serum alpha-fetoprotein solution, weighing serum alpha-fetoprotein 10.0 mg, adding water for dissolution, transferring to a 100mL volumetric flask, and diluting to a scale with distilled water to obtain a 100mL solution;
(3) The magnetic beads nonspecifically adsorb serum alpha fetoprotein: taking 5mL standard serum alpha-fetoprotein solution, adding 5.0 mg magnetic beads prepared in examples 1-6 and comparative example 1 in batches, incubating at 37 ℃ for 3h, performing magnetic separation to remove the magnetic beads, taking 1 mL serum alpha-fetoprotein solution and 5mL Coomassie brilliant blue reagent, uniformly mixing for 8-10 min, scanning in the range of 400-800 nm by using an ultraviolet spectrophotometer, and calculating the amount of nonspecific adsorption protein of the magnetic beads prepared in examples 1-6 and comparative example 1 before and after surface modification.
TABLE 1 adsorption of serum alpha-fetoprotein by magnetic beads (μg/mg)
Table 1 shows the adsorption amount of the surface-modified magnetic beads prepared in examples 1 to 6 to serum alpha-fetoprotein, and it can be seen that the surface-modified magnetic beads prepared in the embodiment of the invention can well inhibit the nonspecific adsorption of proteins on the microsphere surface; the adsorption amount of the surface modified magnetic beads prepared in the example 2 to serum alpha-fetoprotein is smaller than that of the surface modified magnetic beads prepared in the example 1, which shows that the increase of the use amount of 2-hexenal can reduce the non-specific adsorption of the surfaces of the magnetic beads, thereby reducing the adsorption amount to serum alpha-fetoprotein; the adsorption amount of the surface-modified magnetic beads prepared in the example 3 to serum alpha-fetoprotein is smaller than that of the surface-modified magnetic beads prepared in the example 2, which shows that the non-specific adsorption of the protein can be reduced by using hydroxy-1, 4-butanedial to replace 2-hexenal to modify the magnetic beads; the adsorption amount of the surface modified magnetic beads prepared in the example 4 to serum alpha-fetoprotein is smaller than that of the surface modified magnetic beads prepared in the example 3, which shows that the nonspecific adsorption of the magnetic beads can be further reduced by increasing the use amount of the hydroxy-1, 4-butanedialdehyde; the adsorption capacity of the surface modified magnetic beads prepared in the example 5 to serum alpha-fetoprotein is smaller than that of the example 4, which shows that the hydroxyl-1, 4-butanedial and 2-hexenal are used simultaneously, and the two aldehyde compounds can complement each other to generate a synergistic effect, and the non-specific adsorption capacity is further reduced by mixing the modified magnetic beads; the adsorption amount of the surface modified magnetic beads prepared in example 6 to serum alpha-fetoprotein is smaller than that of example 5, which shows that the specific adsorption of the magnetic beads to proteins can be continuously reduced by increasing the use amount of hydroxy-1, 4-butanedialdehyde and 2-hexenal.
3. Preparation of immunomagnetic beads detection chitinase 3-like protein 1 (CHI 3L 1)
3.1, Samples and methods
In order to evaluate the practical application of the magnetic beads, a sandwich type chemiluminescent immunoassay method for quantitatively detecting CHI3L1 antigen by using an azlactone-labeled CHI3L1 antibody is established. First, streptavidin was labeled on the magnetic beads by the active ester method, and then the biotin-labeled CHI3L1 antibody was bound to the magnetic beads by using strong interaction between streptavidin and biotin, and the magnetic beads prepared in examples 1 to 6 and comparative example 1 were added in portions to specifically bind to a certain CHI3L1 antigen. Finally, the antigen-antibody complex-linked magnetic beads are reacted with the azetidine-labeled CHI3L1 antibody to detect the antigen to be detected in the sample.
(1) Streptavidin modified magnetic bead
The beads prepared in examples 1-6 and comparative example 1 were added in portions of 20.0 mg to a centrifuge tube, 10 mL pH =5.5 MES buffer solution was added, the mixture was sonicated uniformly, 80.0 mg EDC and 120.0 mg NHS were added to dissolve the beads, 30 min was activated at 37 ℃, magnetic separation was performed, 10 mL pH =7.2 PBS buffer solution containing 1.0 mg streptavidin was slowly dropped into the beads, shaking reaction was performed at 37 ℃ for 12 h, BSA and glycine solution with a concentration of 0.2% were added to the system to sufficiently block the vacant sites on the beads, and the mixture was washed and stored at 4 ℃ for standby.
(2) CHI3L1 monoclonal antibody labeled biotin
Firstly, adding 2.0 mg biotin into 1 mL ultrapure water for dissolution, then taking 100 mu L of the solution into a centrifuge tube, adding 2.0 mg CHI3L1 monoclonal antibody, incubating the solution on a mixer at 37 ℃ for 3 h, dialyzing the solution after the reaction by using 0.01 mol/L PBS solution with pH=7.2 to remove excessive biotin, adding glycerol with equal amount, and preserving the solution at-20 ℃.
(3) Azate-labeled CHI3L1 monoclonal antibody
First, 0.5 mg azlactone was dissolved well in 500. Mu.L of DMSO, then 500. Mu.L of 0.05 mol/L pH=7.2 PBS solution containing 1.0 mg of CHI3L1 monoclonal antibody was added, 3. 3 h was incubated on a mixer at 37℃and then excess azlactone was removed by dialysis against 0.01 mol/L pH=7.2 PBS solution, and the same amount of glycerol was added and stored at-20 ℃.
(4) Arrangement of excitation liquid
Excitation liquid 1: 0.2 mol/L nitric acid solution containing 0.5% hydrogen peroxide.
Excitation liquid 2: 0.5 mol/L sodium hydroxide solution containing 1% Tween-20.
After the excitation liquid is prepared, the excitation liquid is stored at 2-30 ℃ in a dark place.
(5) Double antibody sandwich method for measuring CHI3L1 content
A CHI3L1 standard at a concentration of 0.225 ng/mL was configured to evaluate the nonspecific binding capacity of the magnetic beads prepared in examples 1-6 and comparative example 1. Adding 100 mu L of biotinylated CHI3L1 antibody and 30 mu L of CHI3L1 standard substance into the microwells, and reacting at 37 ℃ for 20 min to form a biotinylated CHI3L1 antibody-CHI 3L1 antigen complex; 100. Mu.L of streptavidin-modified magnetic beads prepared in examples 1-6 and comparative example 1 were added, respectively, and reacted at 37℃for 20 min, followed by washing with magnetic separation to remove unreacted materials; mu.L of the azlocylate labeled CHI3L1 secondary antibody was added, incubated at 37℃for 20 min to give a double antibody sandwich complex, washed three times with 0.01 mol/L of PBS solution with pH=7.2, and 100. Mu.L of excitation solutions 1 and 2 were added to measure the luminescence value generated by a chemiluminescent detector.
TABLE 2 specific binding of magnetic beads to CHI3L1
Table 2 shows that the specific binding amounts of the immunomagnetic beads prepared in examples 1 to 6 and comparative example 1 to chitosanase 3-like protein 1 (CHI 3L 1) were compared by using an azdine chemiluminescence method, and that the specific adsorption of the surface-modified magnetic beads prepared in examples 1 to 6 to chitosanase 3-like protein 1 (CHI 3L 1) was larger than that of comparative example 1, indicating that the immunomagnetic beads prepared in the embodiment of the present invention can bind more CHI3L1, improving the detection sensitivity to chitosanase 3-like protein 1 (CHI 3L 1); the surface-modified magnetic beads prepared in example 2 have a greater amount of binding to CHI3L1 than example 1, indicating that increasing the amount of 2-hexenal used increases the binding of immunomagnetic beads to CHI3L 1; the amount of the immunomagnetic beads prepared in example 3 bound to CHI3L1 was greater than that of example 2, demonstrating that the use of hydroxy-1, 4-butanedial instead of 2-hexenal to modify the magnetic beads can further enhance the binding of immunomagnetic beads to CHI3L 1; the amount of the immunomagnetic beads prepared in example 4 bound to CHI3L1 was greater than that of example 3, indicating that the amount of CHI3L1 bound could be further increased by increasing the amount of hydroxy-1, 4-butanedial used; the amount of the immunomagnetic beads prepared in example 5 bound to CHI3L1 is greater than that of example 4, which shows that the simultaneous use of hydroxy-1, 4-butanedial and 2-hexenal, which may complement each other, produces a synergistic effect, and can further enhance the binding of the immunomagnetic beads to CHI3L 1; the amount of the immunomagnetic beads prepared in example 6 bound to the CHI3L1 antibody was greater than that of example 5, indicating that increasing the amounts of hydroxy-1, 4-butanedial and 2-hexenal used continued to increase the amount of the immunomagnetic beads bound to the CHI3L1 antibody.
The above examples and/or embodiments are merely for illustrating the preferred embodiments and/or implementations of the present technology, and are not intended to limit the embodiments and implementations of the present technology in any way, and any person skilled in the art should be able to make some changes or modifications to the embodiments and/or implementations without departing from the scope of the technical means disclosed in the present disclosure, and it should be considered that the embodiments and implementations are substantially the same as the present technology.
Claims (7)
1. A method for preparing surface-modified magnetic beads comprises providing nanoscale Fe 3O4 magnetic beads; the nano-scale Fe 3O4 magnetic beads react with a silane coupling agent to carry an amino group to obtain amino-modified Fe 3O4 magnetic beads; adding amino-modified Fe 3O4 magnetic beads into an aldehyde compound solution for reaction, performing magnetic separation, and washing to obtain surface-modified magnetic beads; the silane coupling agent comprises 3-aminopropyl trimethoxy silane; the aldehyde compound solution comprises a 2-hexenal solution and a hydroxy-1, 4-butanedial solution;
The dosage ratio of the Fe 3O4 magnetic beads for modifying amino, the hydroxy-1, 4-butanedial solution and the 2-hexenal solution is 20-100 mg:10-20 mL:10-20 mL; the mass fraction of the hydroxy-1, 4-butanedial solution is 20-30%; the mass fraction of the 2-hexenal solution is 20-30%.
2. The method for preparing surface-modified magnetic beads according to claim 1, wherein the ratio of the amount of the nano-sized Fe 3O4 magnetic beads to the amount of 3-aminopropyl trimethoxysilane is 0.2-0.6 g:40-50: 50 mL.
3. The method for preparing surface-modified magnetic beads according to claim 1, wherein the reaction conditions of the nano-scale Fe 3O4 magnetic beads and the silane coupling agent are stirring speed of 200-400 r/min and reaction speed of 8-12 h at 60-70 ℃.
4. The method for preparing surface-modified magnetic beads according to claim 1, wherein the condition for the reaction of the amino-modified Fe 3O4 magnetic beads in the aldehyde compound solution is that the reaction is carried out at room temperature of 4-8 h.
5. A surface-modified magnetic bead prepared by the method for preparing a surface-modified magnetic bead according to any one of claims 1 to 4.
6. An immunomagnetic bead comprising an immunoligand and a surface-modified magnetic bead produced by the method of any one of claims 1-4 or the surface-modified magnetic bead of claim 5, said immunoligand being bound to said surface-modified magnetic bead, said immunoligand comprising a chitinase 3-like protein 1 polyclonal antibody.
7. A kit for liver disease detection, wherein the reagent in the kit for liver disease detection comprises the surface-modified magnetic bead of claim 5 or the immunomagnetic bead of claim 6.
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