WO2017206714A1 - Method for preparing immunomagnetic nanoparticles - Google Patents

Method for preparing immunomagnetic nanoparticles Download PDF

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WO2017206714A1
WO2017206714A1 PCT/CN2017/084745 CN2017084745W WO2017206714A1 WO 2017206714 A1 WO2017206714 A1 WO 2017206714A1 CN 2017084745 W CN2017084745 W CN 2017084745W WO 2017206714 A1 WO2017206714 A1 WO 2017206714A1
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magnetic particles
preparation
magnetic
coated
nano
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PCT/CN2017/084745
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郑招荣
刘金超
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深圳市瀚德标检生物工程有限公司
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • 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
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/01—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials
    • H01F1/03—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity
    • H01F1/12—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of soft-magnetic materials
    • H01F1/34—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of soft-magnetic materials non-metallic substances, e.g. ferrites
    • H01F1/342—Oxides
    • H01F1/344—Ferrites, e.g. having a cubic spinel structure (X2+O)(Y23+O3), e.g. magnetite Fe3O4
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/01—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials
    • H01F1/03—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity
    • H01F1/12—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of soft-magnetic materials
    • H01F1/34—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of soft-magnetic materials non-metallic substances, e.g. ferrites
    • H01F1/36—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of soft-magnetic materials non-metallic substances, e.g. ferrites in the form of particles
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F41/00—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties
    • H01F41/02—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties for manufacturing cores, coils, or magnets

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for preparing immunomagnetic nanoparticles.
  • the ferroferric oxide nanoparticles have good biocompatibility and magnetic properties. They can be rapidly aggregated and separated from other components under the action of an external magnetic field, so that they can be sorted and labeled in the cell, and the protein is separated and purified, and the drug is transported. Magnetic resonance imaging, cell labeling, targeted drug carriers, etc. are widely used; however, the triiron tetroxide nanoparticles without surface modification have large surface activation energy and large intergranular magnetostatic properties in solution due to their small particle size. Interacting with the galvanic moment results in poor stability, easy agglomeration, deposition, and easy oxidation. It is therefore necessary to modify it to change its surface properties to prevent it from being oxidized and better dispersed in the aqueous phase. At the same time, the surface of the modified high-density active group can be coupled with biomolecules, cell surface and other active molecules to meet different applications.
  • the invention patents of CNI01241130A and CN1667413A are structures for coating an organic polymer such as polystyrene, and the invention patents of CNA015196482B and CN1872028A, respectively, are prepared by modifying and activating carboxyl groups to prepare immunomagnetic particles.
  • Publication No. CN 103357359 A uses a Schiff reaction to form an imine to couple magnetic nanoparticles with biologically active molecules.
  • Publication No. CN 102766191 B is coupled to a protein by multiple modification and group protection on the surface of the magnetic particles.
  • the problems in the preparation technology of these magnetic particles are high process conditions, cumbersome preparation process, involving organic toxic substances, large-scale equipment, poor repeatability between batches, difficulty in large-scale stable preparation, low coupling efficiency of biologically active molecules, and easy It falls off, etc., so it is greatly limited in industrial applications.
  • the technical problem to be solved by the present invention is to overcome the technical bottlenecks such as high requirements of the prior art process conditions, cumbersome preparation process, involving organic toxic substances, difficulty in large-scale stable preparation, low coupling efficiency, poor stability, and easy falling off.
  • a preparation process of immunomagnetic nanoparticles with high coupling efficiency, uniform particle size, good stability, simple process, short cycle, low toxicity, low cost and no need for large equipment was proposed.
  • the present invention discloses a method for preparing immunomagnetic nanoparticles, the method comprising the following steps:
  • the nano magnetic particles prepared in the step a are magnetically separated, and the suspension is washed with an alkali solution and redispersed in an alkali solution; the ethyl orthosilicate is dissolved in ethanol, added to the dispersion, and stirred, on the surface of the magnetic particles.
  • the resulting layer of silicon dioxide is coated and magnetically separated to obtain surface silicon coated magnetic particles;
  • the carboxyl magnetic particles prepared in step c are washed and dispersed in a buffer, activated by Sulfo-NHS and EDC, magnetically separated, and added with an antigen, an antibody or other biologically active molecule, and oscillated and magnetically separated to obtain immunomagnetic nanoparticles.
  • the method steps are as follows:
  • the nano magnetic particles prepared in the step a are magnetically separated, and the suspension is washed three times with an alkali solution having a pH of 10 to 13, redispersed in an alkali solution, and the pH is adjusted to 10 to 13, and the tetraethyl orthosilicate is dissolved.
  • an alkali solution having a pH of 10 to 13, redispersed in an alkali solution, and the pH is adjusted to 10 to 13, and the tetraethyl orthosilicate is dissolved.
  • the mixture is stirred at 30 to 50 ° C for 4 to 5 hours, and a layer of silica formed on the surface of the magnetic particles is coated; magnetic separation is obtained by surface silicon coating.
  • the aminated silane and the acid anhydride are stirred in an anhydrous solvent for 10 to 60 minutes in a molar ratio of the acid anhydride group to the amino group (1.2 to 2): 1, to obtain a carboxylated silane.
  • the carboxylated silane is dissolved in absolute ethanol, and the nano magnetic particles prepared in 1.2 are added, and the pH is adjusted to 10 to 13 with an alkali solution, stirred at 30 to 50 ° C for 4 to 5 hours, and then washed with absolute ethanol and purified water. Magnetic separation to obtain nano-magnetic particles modified with surface carboxyl groups;
  • the carboxyl magnetic particles prepared in the step c are washed three times with MES buffer having a pH of 6.0 to 7.0, and then dispersed in the buffer, and the oscillation reaction is activated by using Sulfo-NHS and EDC at room temperature for 0.5 to 1 hour. , magnetic separation, washing to remove unreacted reagents, adding antigen, antibody, streptavidin molecules, shaking reaction at room temperature for 3 h, magnetic separation, buffer washed.
  • the amino group includes a primary amino group and a secondary amino group.
  • the divalent iron salt is one of ferrous sulfate and ferrous chloride;
  • the ferric salt is one of ferric chloride, ferric sulfate, ferric nitrate and ferric citrate;
  • the alkali solution is ammonia water. Or one of tetraaminoammonium hydroxide.
  • the molar ratio of the divalent iron salt is 0.01 mol/L to 1 mol/L; the molar concentration of the ferric salt is 0.01 mol/L to 2 mol/L; and the concentration of hydrochloric acid is 0.1 mol/L to 2 mol/L.
  • the molar ratio of the divalent iron salt, the trivalent iron salt to the lye is (1 to 2): (1 to 2): (4 to 20).
  • the mass ratio of the tetraethyl orthosilicate to the ferric oxide is (1 to 5): (1 to 20).
  • the acid anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, maleic anhydride, phthalic anhydride;
  • the aminated silane is ⁇ -aminopropyltriethoxysilane, ⁇ - Aminopropyltrimethoxysilane, diethylenetriaminopropyltrimethoxysilane, N-( ⁇ -aminoethyl)- ⁇ -aminopropyltriethyl
  • anhydrous solvent is N,N-dimethylformamide, dimethyl sulfoxide One kind.
  • the molar ratio of the acid anhydride to the carboxylated silane is (1 to 5): (1 to 20).
  • the mass ratio of the magnetic nanoparticles to EDC, Sulfo-NHS is 1 g: (1 to 100 mmol): (1 to 50 mmol); the mass ratio of the magnetic particles to the antibody is 1 g: (0.1) ⁇ 10mg).
  • the carboxyl group-modified magnetic nanoparticle of the present invention comprises a ferroferric oxide core, a silica shell layer and a surface-modified active group carboxyl group, and the carboxyl group can be coupled to an antibody, an antigen, and a half required for in vitro diagnosis by activation.
  • Biologically active molecules such as antigen, avidin, streptavidin, etc., due to the high density of carboxyl groups modified on the surface of magnetic particles, the coupling efficiency is higher, and the analyte can be quickly and efficiently separated from other molecules by an external magnetic field, so The fields of diagnosis, microbial isolation, cell labeling, and targeted drugs have broad application prospects.
  • the prepared magnetic particles have uniform particle size, good stability and adjustable size, which can meet different needs. There is no sedimentation after storage for more than half a year at room temperature.
  • EXAMPLE 1 This example discloses a method for preparing an immunomagnetic nanoparticle (avidin/streptavidin-coated immunomagnetic nanoparticle), the method comprising the following steps:
  • ferromagnetic ferromagnetic microparticle core Weigh 40mmol FeCl 2 and dissolve it in 4mL purified water. Pour the mixed solution into 250ml containing 1mmol/L sodium citrate, 0.8mol/L sodium nitrate, 0.1mol/L. In sodium hydroxide, vigorously stir at 70 ° C for 60 min, cool to room temperature, wash twice with 1% TMAH, magnetically separate, discard the supernatant. The pellet was washed 2 to 3 times with 0.1% TMAH. Resuspend in 25 mL of 0.1% TMAH.
  • Example 2 This example discloses a method for preparing immunomagnetic nanoparticles (rabbit anti-FITC antibody-modified immunomagnetic nanoparticles), the method comprising the following steps:
  • Magnetic particles modified with surface carboxyl groups 15 mmol of succinic anhydride and 10 mmol of ⁇ -aminopropyltriethoxysilane were dissolved in 1 ml of anhydrous DMF and reacted at 40 ° C for 30 min, and the mixture was dissolved in 20 ml of ethanol.

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Abstract

A method for preparing immunomagnetic nanoparticles. Surfaces of ferroferric oxide magnetic nanoparticles are coated with silicate ester to obtain silicon-coated magnetic nanoparticles; the surface activation energy of the magnetic nanoparticles is reduced, so that the stability thereof is greatly improved; in addition, the concentration coating thickness of the silicate ester can be adjusted. Afterwards, the surfaces of the magnetic nanoparticles are modified with carboxyl, and the carboxyl-modified magnetic nanoparticles are activated by means of an activating agent and then covalently bound to free amino groups of bioactive molecules by means of amido bonds; the covalent binding is firmer than non-covalent binding between an imine structure obtained by aldehyde-amine condensation and colloidal gold. In the whole process, the reaction conditions are mild, no inert gas environment, no severe condition, such as high temperature and high pressure, no complicated device and large-sized apparatus are needed, raw materials are readily available, and there is no need of a large quantity of organic solvents. In addition, the synthesized magnetic nanoparticles are adjustable in particle diameter.

Description

一种免疫磁性纳米微粒的制备方法Preparation method of immunomagnetic nano particles 技术领域Technical field
本发明属于生物技术领域，具体涉及一种免疫磁性纳米微粒的制备方法。The invention belongs to the field of biotechnology, and in particular relates to a method for preparing immunomagnetic nanoparticles.
背景技术Background technique
四氧化三铁纳米微粒具有良好的生物相容性和磁学性能，其在外加磁场作用下，可迅速聚集并与其它组分分离，使其在细胞分选和标记，蛋白质分离纯化，药物输送，磁共振造影、细胞标记、靶向药物载体等方面得到广泛应用；然而，未经表面修饰处理的四氧化三铁纳米微粒在溶液中由于其粒径小，表面活化能大，颗粒间静磁与电偶矩相互作用，导致其稳定性很差，容易团聚，产生沉积，同时还容易被氧化。因此需要对其进行修饰，改变其表面性质，防止其被氧化并能够更好地分散在水相中。同时其表面经修饰的高密度活性基团能够与生物分子、细胞表面以及其它活性分子进行偶联以满足不同的应用。The ferroferric oxide nanoparticles have good biocompatibility and magnetic properties. They can be rapidly aggregated and separated from other components under the action of an external magnetic field, so that they can be sorted and labeled in the cell, and the protein is separated and purified, and the drug is transported. Magnetic resonance imaging, cell labeling, targeted drug carriers, etc. are widely used; however, the triiron tetroxide nanoparticles without surface modification have large surface activation energy and large intergranular magnetostatic properties in solution due to their small particle size. Interacting with the galvanic moment results in poor stability, easy agglomeration, deposition, and easy oxidation. It is therefore necessary to modify it to change its surface properties to prevent it from being oxidized and better dispersed in the aqueous phase. At the same time, the surface of the modified high-density active group can be coupled with biomolecules, cell surface and other active molecules to meet different applications.
目前已经有多种四氧化三铁纳米微粒的制备、表面修饰以及偶联生物分子的方法。公开号分别为CNI01241130A、CN1667413A的发明专利为包覆聚苯乙烯等有机高分子聚合物的结构，公开号分别为CNI015196482B和CN1872028A的发明专利是通过修饰和活化羧基制备出免疫磁性微粒。公开号为CN 103357359 A利用Schiff反应生成亚胺来偶联磁纳米微粒与生物活性分子，公开号为CN 102766191 B是通过在磁微粒表面多次修饰与基团保护从而与蛋白质偶联。目前这些磁微粒的制备技术存在的问题是工艺条件要求较高、制备过程繁琐、涉及有机毒性物质、需要大型仪器设备、批间重复性差、难以大量稳定制备、生物活性分子偶联效率低、容易脱落等，因此在工业化应用上受到较大限制。 There are a variety of methods for the preparation, surface modification, and coupling of biomolecules of ferroferric oxide nanoparticles. The invention patents of CNI01241130A and CN1667413A are structures for coating an organic polymer such as polystyrene, and the invention patents of CNA015196482B and CN1872028A, respectively, are prepared by modifying and activating carboxyl groups to prepare immunomagnetic particles. Publication No. CN 103357359 A uses a Schiff reaction to form an imine to couple magnetic nanoparticles with biologically active molecules. Publication No. CN 102766191 B is coupled to a protein by multiple modification and group protection on the surface of the magnetic particles. At present, the problems in the preparation technology of these magnetic particles are high process conditions, cumbersome preparation process, involving organic toxic substances, large-scale equipment, poor repeatability between batches, difficulty in large-scale stable preparation, low coupling efficiency of biologically active molecules, and easy It falls off, etc., so it is greatly limited in industrial applications.
发明内容Summary of the invention
为此，本发明所要解决的技术问题在于克服现有技术工艺条件要求较高、制备过程繁琐、涉及有机毒性物质、难以大量稳定制备、偶联效率低、稳定性差、易脱落等技术瓶颈，从而提出一种偶联效率高、粒径均匀、稳定性好、工艺简单、周期短、低毒、低成本、无需大型设备的免疫磁性纳米微粒的制备工艺。Therefore, the technical problem to be solved by the present invention is to overcome the technical bottlenecks such as high requirements of the prior art process conditions, cumbersome preparation process, involving organic toxic substances, difficulty in large-scale stable preparation, low coupling efficiency, poor stability, and easy falling off. A preparation process of immunomagnetic nanoparticles with high coupling efficiency, uniform particle size, good stability, simple process, short cycle, low toxicity, low cost and no need for large equipment was proposed.
为解决上述技术问题，本发明公开了一种免疫磁性纳米微粒的制备方法，所述方法包括如下步骤：In order to solve the above technical problems, the present invention discloses a method for preparing immunomagnetic nanoparticles, the method comprising the following steps:
a.制备四氧化三铁纳米微粒内核a. Preparation of ferroferric oxide nanoparticle core
配制二价铁盐溶液，然后加至含柠檬酸钠以及高浓度硝酸盐的碱性溶液中搅拌；然后进行沉淀离心分离，得到磁悬浮液；Preparing a divalent iron salt solution, and then adding it to an alkaline solution containing sodium citrate and a high concentration of nitrate; then performing centrifugation and centrifugation to obtain a magnetic suspension;
b.制备表面硅包覆的纳米磁微粒b. Preparation of surface silicon coated nano magnetic particles
用步骤a制备的纳米磁微粒，磁性分离，用碱液洗涤悬液，重分散于碱液中；将正硅酸乙酯溶解于乙醇中，并加入至分散液中，搅拌，在磁微粒表面生成的一层二氧化硅得到的包覆，磁分离得到表面硅包覆的磁微粒；The nano magnetic particles prepared in the step a are magnetically separated, and the suspension is washed with an alkali solution and redispersed in an alkali solution; the ethyl orthosilicate is dissolved in ethanol, added to the dispersion, and stirred, on the surface of the magnetic particles. The resulting layer of silicon dioxide is coated and magnetically separated to obtain surface silicon coated magnetic particles;
c.制备表面羧基修饰的磁微粒c. Preparation of surface carboxyl modified magnetic particles
称取氨基化硅烷与酸酐，在无水溶剂中搅拌，得到羧基化硅烷；将羧基化硅烷溶解于无水乙醇中，加入纳米磁微粒，加碱液，并搅拌，然后用无水乙醇与纯化水洗涤，磁分离，得到表面羧基修饰的纳米磁微粒；Weighing the aminated silane and the acid anhydride, stirring in an anhydrous solvent to obtain a carboxylated silane; dissolving the carboxylated silane in absolute ethanol, adding the nano magnetic particles, adding the alkali solution, stirring, and then purifying with absolute ethanol Water washing, magnetic separation, obtaining nano-magnetic particles modified with surface carboxyl groups;
d.制备免疫磁微粒d. Preparation of immunomagnetic particles
将步骤c制备的羧基磁微粒洗涤分散于缓冲液中，用Sulfo-NHS与EDC活化反应后，磁分离，加入抗原、抗体或其它生物活性分子，振荡反应，磁分离即得免疫磁性纳米微粒。The carboxyl magnetic particles prepared in step c are washed and dispersed in a buffer, activated by Sulfo-NHS and EDC, magnetically separated, and added with an antigen, an antibody or other biologically active molecule, and oscillated and magnetically separated to obtain immunomagnetic nanoparticles.
优选的，所述方法步骤如下：Preferably, the method steps are as follows:
a.制备四氧化三铁纳米微粒内核a. Preparation of ferroferric oxide nanoparticle core
配制二价铁盐溶液，加至剧烈搅拌的含柠檬酸钠以及高浓度硝酸盐的碱性溶液中，在25～90℃条件下搅拌5分钟以上，得到稳定的磁悬浮液； Preparing a divalent iron salt solution, adding to a vigorously stirred alkaline solution containing sodium citrate and a high concentration of nitrate, stirring at 25 to 90 ° C for more than 5 minutes to obtain a stable magnetic suspension;
b.制备表面硅包覆的纳米磁微粒b. Preparation of surface silicon coated nano magnetic particles
用步骤a制备的纳米磁微粒，磁性分离，用pH值为10～13的碱液洗涤悬液3次，重分散于碱液中，调节pH值为10～13，将正硅酸乙酯溶解于乙醇中，并按比例加入至分散液中，30～50℃条件下混合物搅拌4～5小时，在磁微粒表面生成的一层二氧化硅得到的包覆；磁分离得到表面硅包覆的磁微粒；The nano magnetic particles prepared in the step a are magnetically separated, and the suspension is washed three times with an alkali solution having a pH of 10 to 13, redispersed in an alkali solution, and the pH is adjusted to 10 to 13, and the tetraethyl orthosilicate is dissolved. In ethanol, and added to the dispersion in proportion, the mixture is stirred at 30 to 50 ° C for 4 to 5 hours, and a layer of silica formed on the surface of the magnetic particles is coated; magnetic separation is obtained by surface silicon coating. Magnetic particle
c.制备表面羧基修饰的磁微粒c. Preparation of surface carboxyl modified magnetic particles
按酸酐基团与氨基摩尔比(1.2～2)∶1将称取氨基化硅烷与酸酐在无水溶剂中搅拌10到60分钟，得到羧基化硅烷。将羧基化硅烷溶解于无水乙醇中，加入1.2制备的纳米磁微粒，用碱液调节pH值至10～13，30～50℃搅拌4～5小时，然后用无水乙醇与纯化水洗涤，磁分离，得到表面羧基修饰的纳米磁微粒；The aminated silane and the acid anhydride are stirred in an anhydrous solvent for 10 to 60 minutes in a molar ratio of the acid anhydride group to the amino group (1.2 to 2): 1, to obtain a carboxylated silane. The carboxylated silane is dissolved in absolute ethanol, and the nano magnetic particles prepared in 1.2 are added, and the pH is adjusted to 10 to 13 with an alkali solution, stirred at 30 to 50 ° C for 4 to 5 hours, and then washed with absolute ethanol and purified water. Magnetic separation to obtain nano-magnetic particles modified with surface carboxyl groups;
d.制备免疫磁微粒d. Preparation of immunomagnetic particles
优选的，将步骤c制备的羧基磁微粒用pH值为6.0～7.0的MES缓冲液洗涤三次，再分散于此缓冲液中，用Sulfo-NHS与EDC在室温下活化振荡反应0.5～1小时后，磁分离，洗涤除去未反应的试剂，加入抗原、抗体、链霉亲和素分子，室温振荡反应3h，磁分离，缓冲液洗涤后即得。Preferably, the carboxyl magnetic particles prepared in the step c are washed three times with MES buffer having a pH of 6.0 to 7.0, and then dispersed in the buffer, and the oscillation reaction is activated by using Sulfo-NHS and EDC at room temperature for 0.5 to 1 hour. , magnetic separation, washing to remove unreacted reagents, adding antigen, antibody, streptavidin molecules, shaking reaction at room temperature for 3 h, magnetic separation, buffer washed.
优选的，所述氨基包括伯胺基和仲胺基。Preferably, the amino group includes a primary amino group and a secondary amino group.
优选的，所述二价铁盐为硫酸亚铁、氯化亚铁中的一种；三价铁盐为氯化铁、硫酸铁、硝酸铁、柠檬酸铁中的一种；碱液为氨水或四氨基氢氧化铵中的一种。Preferably, the divalent iron salt is one of ferrous sulfate and ferrous chloride; the ferric salt is one of ferric chloride, ferric sulfate, ferric nitrate and ferric citrate; the alkali solution is ammonia water. Or one of tetraaminoammonium hydroxide.
优选的，所述二价铁盐摩尔浓度比为0.01mol/L～1mol/L；三价铁盐的摩尔浓度为0.01mol/L～2mol/L；盐酸浓度为0.1mol/L～2mol/L；二价铁盐、三价铁盐与碱液的摩尔比为(1～2)∶(1～2)∶(4～20)。Preferably, the molar ratio of the divalent iron salt is 0.01 mol/L to 1 mol/L; the molar concentration of the ferric salt is 0.01 mol/L to 2 mol/L; and the concentration of hydrochloric acid is 0.1 mol/L to 2 mol/L. The molar ratio of the divalent iron salt, the trivalent iron salt to the lye is (1 to 2): (1 to 2): (4 to 20).
优选的，所述正硅酸乙酯与四氧化三铁的质量比为(1～5)∶(1～20)。Preferably, the mass ratio of the tetraethyl orthosilicate to the ferric oxide is (1 to 5): (1 to 20).
优选的，所述酸酐为丁二酸酐、戊二酸酐、己二酸酐、马来酸酐、邻苯二甲酸酐中的一种；氨基化硅烷为γ-氨丙基三乙氧基硅烷、γ-氨丙基三甲氧基硅烷、二乙烯三氨基丙基三甲氧基硅烷、N-(β-氨乙基)-γ-氨丙基三乙 氧基硅烷、N-(β-氨乙基)-γ-氨丙基甲基二甲氧基硅烷中的一种；无水溶剂为N，N-二甲基甲酰胺、二甲亚砜中的一种。Preferably, the acid anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, maleic anhydride, phthalic anhydride; the aminated silane is γ-aminopropyltriethoxysilane, γ- Aminopropyltrimethoxysilane, diethylenetriaminopropyltrimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltriethyl One of oxysilane, N-(β-aminoethyl)-γ-aminopropylmethyldimethoxysilane; anhydrous solvent is N,N-dimethylformamide, dimethyl sulfoxide One kind.
优选的，所述酸酐与羧基化硅烷的摩尔比为(1～5)∶(1～20)。Preferably, the molar ratio of the acid anhydride to the carboxylated silane is (1 to 5): (1 to 20).
更为优选的，所述磁纳米微粒与EDC，Sulfo-NHS的质量摩尔比为1g∶(1～100mmol)∶(1～50mmol)；加入的所述磁微粒与抗体的质量比1g∶(0.1～10mg)。More preferably, the mass ratio of the magnetic nanoparticles to EDC, Sulfo-NHS is 1 g: (1 to 100 mmol): (1 to 50 mmol); the mass ratio of the magnetic particles to the antibody is 1 g: (0.1) ~10mg).
本发明的上述技术方案相比现有技术具有以下优点：The above technical solution of the present invention has the following advantages over the prior art:
(1)本发明的羧基修饰的磁纳米微粒，包括四氧化三铁内核，二氧化硅壳层以及表面修饰的活性基团羧基，羧基通过活化可偶联体外诊断所需的抗体、抗原、半抗原、亲和素、链霉亲和素等生物活性分子，由于磁微粒表面修饰的羧基密度大，偶联效率更高，可通过外磁场将待测物与其它分子快速有效分离，因此在体外诊断、微生物分离、细胞标记、靶向药物等领域具有着广泛的应用前景。(1) The carboxyl group-modified magnetic nanoparticle of the present invention comprises a ferroferric oxide core, a silica shell layer and a surface-modified active group carboxyl group, and the carboxyl group can be coupled to an antibody, an antigen, and a half required for in vitro diagnosis by activation. Biologically active molecules such as antigen, avidin, streptavidin, etc., due to the high density of carboxyl groups modified on the surface of magnetic particles, the coupling efficiency is higher, and the analyte can be quickly and efficiently separated from other molecules by an external magnetic field, so The fields of diagnosis, microbial isolation, cell labeling, and targeted drugs have broad application prospects.
(2)制得的磁微粒粒径均一、稳定好、大小可调节，可满足不同需求，室温下存放半年以上均无沉降。(2) The prepared magnetic particles have uniform particle size, good stability and adjustable size, which can meet different needs. There is no sedimentation after storage for more than half a year at room temperature.
(3)制备工艺简单易行、周期短，所有原物料均低毒、价廉、易得，无需大型设备与复杂装置，易于大规模制备。(3) The preparation process is simple and easy, the cycle is short, all the raw materials are low-toxic, inexpensive, and easy to obtain, and large-scale equipment and complicated devices are not needed, and it is easy to prepare on a large scale.
具体实施方式detailed description
实施例1  本实施例公开了一种免疫磁性纳米微粒(亲和素/链霉亲合素包被的免疫磁纳米微粒)的制备方法，所述方法包括如下步骤：EXAMPLE 1 This example discloses a method for preparing an immunomagnetic nanoparticle (avidin/streptavidin-coated immunomagnetic nanoparticle), the method comprising the following steps:
(1)四氧化三铁磁微粒内核的制备：称取40mmol FeCl2，溶于4mL纯化水中，将混合溶液倒入250ml含1mmol/L柠檬酸钠、0.8mol/L硝酸钠、0.1mol/L氢氧化钠中，70℃条件下剧烈搅拌60min，冷却至室温，用1％TMAH洗涤两次，磁力分离，弃上清。沉淀用0.1％ TMAH洗涤2～3次。重悬于25mL 0.1％TMAH中。(1) Preparation of ferromagnetic ferromagnetic microparticle core: Weigh 40mmol FeCl 2 and dissolve it in 4mL purified water. Pour the mixed solution into 250ml containing 1mmol/L sodium citrate, 0.8mol/L sodium nitrate, 0.1mol/L. In sodium hydroxide, vigorously stir at 70 ° C for 60 min, cool to room temperature, wash twice with 1% TMAH, magnetically separate, discard the supernatant. The pellet was washed 2 to 3 times with 0.1% TMAH. Resuspend in 25 mL of 0.1% TMAH.
(2)表面二氧化硅修饰的磁微粒：取25mL(1)制备的磁微粒内核，加 入3ml 10％的正硅酸乙酯的乙醇溶液，室温下剧烈搅拌3小时，0.1％TMAH洗涤2次，即得表面二氧化硅包覆的磁纳米微粒(2) Surface silica modified magnetic particles: take 25mL (1) prepared magnetic particle core, plus Add 3ml of 10% ethyl orthosilicate in ethanol, stir vigorously for 3 hours at room temperature, and wash 0.1% TMAH twice to obtain surface silica coated magnetic nanoparticles.
(3)表面羧基修饰的磁微粒：称取25mmol丁二酸酐，10mmol N-(β-氨乙基)-γ-氨丙基甲基二甲氧基硅烷溶解于1ml无水DMF中室温振荡反应30min，然后将混合物溶于20ml乙醇中，与(2)制备的二氧化硅包覆的磁微粒混合，TMAH调节pH至12.0，50℃条件下搅拌5小时，冷却至室温，磁分离，用丙酮与纯化水洗涤除去未结合物，最后分散于纯化水中(3) Magnetic particles modified with surface carboxyl groups: 25 mmol of succinic anhydride was weighed, and 10 mmol of N-(β-aminoethyl)-γ-aminopropylmethyldimethoxysilane was dissolved in 1 ml of anhydrous DMF and shaken at room temperature. After 30 min, the mixture was dissolved in 20 ml of ethanol, mixed with the silica-coated magnetic particles prepared in (2), the pH was adjusted to 12.0 by TMAH, stirred at 50 ° C for 5 hours, cooled to room temperature, magnetically separated, and acetone was used. Washed with purified water to remove unbound, and finally dispersed in purified water
(4)磁微粒与链霉亲和素偶联：取20ml 10mg/ml羧基修饰磁微粒，加入3mmol EDC，2mmol Sulfo-NHS，室温下振荡反应2小时，磁分离，加入1mg亲和素/链霉亲合素，室温振荡反应4小时以上，磁分离，保留上清液用于测定链霉亲和素的偶联效率。用MES缓冲液洗涤分离物，即得所述的亲和素/链霉亲合素包被的免疫磁纳米微粒。(4) Coupling of magnetic particles with streptavidin: 20 ml of 10 mg/ml carboxyl modified magnetic particles, 3 mmol of EDC, 2 mmol of Sulfo-NHS, shaking at room temperature for 2 hours, magnetic separation, adding 1 mg of avidin/chain Mycorrhizin was shaken at room temperature for more than 4 hours, magnetically separated, and the supernatant was retained for determination of the coupling efficiency of streptavidin. The isolate was washed with MES buffer to obtain the avidin/streptavidin-coated immunomagnetic nanoparticles.
实施例2  本实施例公开了一种免疫磁性纳米微粒(兔抗FITC抗体修饰的免疫磁纳米微粒)的制备方法，所述方法包括如下步骤：Example 2 This example discloses a method for preparing immunomagnetic nanoparticles (rabbit anti-FITC antibody-modified immunomagnetic nanoparticles), the method comprising the following steps:
(1)四氧化三铁磁微粒内核的制备：称取15mmol Fe2(SO4)3，溶解于4mL纯化水中，将混合溶液倒入100ml含0.5mmol/L柠檬酸钠、1mol/L硝酸钾的1％TMAH溶液中，加热至60℃，剧烈搅拌反应30min。冷却至室温，磁性分离，弃上清。用0.1％TMAH洗涤分离物2～3次。重悬于15mL 0.1％ TMAH中，得到稳定的磁悬浮液。(1) Preparation of ferromagnetic ferromagnetic microparticle core: Weigh 15mmol Fe2(SO 4 ) 3 , dissolve it in 4mL purified water, and pour the mixed solution into 100ml containing 0.5mmol/L sodium citrate and 1mol/L potassium nitrate. In a 1% TMAH solution, heat to 60 ° C and stir vigorously for 30 min. Cool to room temperature, magnetically separate, discard the supernatant. The isolate was washed 2 to 3 times with 0.1% TMAH. Resuspend in 15 mL of 0.1% TMAH to give a stable magnetic suspension.
(2)表面二氧化硅修饰的磁微粒：取15mL(1)制备的磁微粒内核，加入3ml 30％的四乙氧基正硅烷的乙醇溶液，室温下剧烈搅拌3小时，1％TMAH洗涤2次，即得表面二氧化硅包覆的磁纳米微粒(2) Surface silica modified magnetic particles: Take 15 mL of the magnetic particle core prepared in (1), add 3 ml of 30% tetraethoxysilane solution in ethanol, stir vigorously for 3 hours at room temperature, 1% TMAH wash 2 Second, the surface of the silica coated magnetic nanoparticles
(3)表面羧基修饰的磁微粒：称取15mmol丁二酸酐、10mmol γ-氨丙基三乙氧基硅烷溶解于1ml无水DMF中40℃条件下反应30min，将混合物溶于20ml乙醇中，与(2)制备的二氧化硅包覆的磁微粒混合，TMAH调节pH至12.0，50℃条件下搅拌5小时，冷却至室温，磁分离，用乙醇洗涤除去未结合的试剂，再纯化水洗涤，重悬于纯化水中。 (3) Magnetic particles modified with surface carboxyl groups: 15 mmol of succinic anhydride and 10 mmol of γ-aminopropyltriethoxysilane were dissolved in 1 ml of anhydrous DMF and reacted at 40 ° C for 30 min, and the mixture was dissolved in 20 ml of ethanol. Mixing with the silica-coated magnetic particles prepared in (2), adjusting the pH to 12.0 with TMAH, stirring at 50 ° C for 5 hours, cooling to room temperature, magnetic separation, washing with ethanol to remove unbound reagent, and then washing with purified water. Resuspended in purified water.
(4)磁微粒与兔抗FITC抗体偶联：移取200mg羧基修饰磁微粒，重分散于pH值为6.5的MES缓冲液中，加入4mmol EDC，2mmol Sulfo-NHS，室温下振荡反应1小时，磁分离，加入1mg兔抗FITC抗体，室温振荡反应4小时，磁分离，保留上清液用于测定兔抗FITC抗体的偶联效率，用MES缓冲液洗涤分离物，即得所述的兔抗FITC抗体包被的免疫磁纳米微粒。(4) Coupling of magnetic particles with rabbit anti-FITC antibody: 200 mg of carboxyl modified magnetic particles were transferred, redispersed in MES buffer at pH 6.5, 4 mmol EDC, 2 mmol Sulfo-NHS was added, and the reaction was shaken at room temperature for 1 hour. Magnetic separation, adding 1 mg rabbit anti-FITC antibody, shaking reaction at room temperature for 4 hours, magnetic separation, retaining the supernatant for determining the coupling efficiency of rabbit anti-FITC antibody, washing the isolate with MES buffer, then obtaining the rabbit anti-antibody FITC antibody coated immunomagnetic nanoparticles.
实验例Experimental example
将实施例1与实施例2所得的免疫纳米微粒的偶联效率与稳定性与现有的技术所得进行对比，得到如下表格：The coupling efficiency and stability of the immuno-nanoparticles obtained in Example 1 and Example 2 were compared with those obtained in the prior art, and the following table was obtained:
偶联效率Coupling efficiency 本实施例This embodiment 醛胺缩合偶联Aldehyde amine condensation coupling 胶体金法偶联Colloidal gold coupling
链霉亲合素偶联磁微粒Streptavidin-coupled magnetic particles 92％92% 81％81% 86％86%
兔抗FITC抗体偶联磁微粒Rabbit anti-FITC antibody-coupled magnetic particles 93％93% 83％83% 86％86%
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。 It is apparent that the above-described embodiments are merely illustrative of the examples, and are not intended to limit the embodiments. Other variations or modifications of the various forms may be made by those skilled in the art in light of the above description. There is no need and no way to exhaust all of the implementations. Obvious changes or variations resulting therefrom are still within the scope of the invention.

Claims (9)

  1. 一种免疫磁性纳米微粒的制备方法，其特征在于，所述方法包括如下步骤：A method for preparing immunomagnetic nanoparticles, characterized in that the method comprises the following steps:
    a.制备四氧化三铁纳米微粒内核a. Preparation of ferroferric oxide nanoparticle core
    配制二价铁盐溶液，然后加至含柠檬酸钠以及高浓度硝酸盐的碱性溶液中搅拌；然后进行沉淀离心分离，得到磁悬浮液；Preparing a divalent iron salt solution, and then adding it to an alkaline solution containing sodium citrate and a high concentration of nitrate; then performing centrifugation and centrifugation to obtain a magnetic suspension;
    b.制备表面硅包覆的纳米磁微粒b. Preparation of surface silicon coated nano magnetic particles
    用步骤a制备的纳米磁微粒，磁性分离，用碱液洗涤悬液，重分散于碱液中；将正硅酸乙酯溶解于乙醇中，并加入至分散液中，搅拌，在磁微粒表面生成的一层二氧化硅得到的包覆，磁分离得到表面硅包覆的磁微粒；The nano magnetic particles prepared in the step a are magnetically separated, and the suspension is washed with an alkali solution and redispersed in an alkali solution; the ethyl orthosilicate is dissolved in ethanol, added to the dispersion, and stirred, on the surface of the magnetic particles. The resulting layer of silicon dioxide is coated and magnetically separated to obtain surface silicon coated magnetic particles;
    c.制备表面羧基修饰的磁微粒c. Preparation of surface carboxyl modified magnetic particles
    称取氨基化硅烷与酸酐，在无水溶剂中搅拌，得到羧基化硅烷；将羧基化硅烷溶解于无水乙醇中，加入纳米磁微粒，加碱液，并搅拌，然后用无水乙醇与纯化水洗涤，磁分离，得到表面羧基修饰的纳米磁微粒；Weighing the aminated silane and the acid anhydride, stirring in an anhydrous solvent to obtain a carboxylated silane; dissolving the carboxylated silane in absolute ethanol, adding the nano magnetic particles, adding the alkali solution, stirring, and then purifying with absolute ethanol Water washing, magnetic separation, obtaining nano-magnetic particles modified with surface carboxyl groups;
    d.制备免疫磁微粒d. Preparation of immunomagnetic particles
    将步骤c制备的羧基磁微粒洗涤分散于缓冲液中，用Sulfo-NHS与EDC活化反应后，磁分离，加入抗原、抗体或其它生物活性分子，振荡反应，磁分离即得免疫磁性纳米微粒。The carboxyl magnetic particles prepared in step c are washed and dispersed in a buffer, activated by Sulfo-NHS and EDC, magnetically separated, and added with an antigen, an antibody or other biologically active molecule, and oscillated and magnetically separated to obtain immunomagnetic nanoparticles.
  2. 如权利要求1所述的制备方法，其特征在于，所述方法步骤如下：The preparation method according to claim 1, wherein the method steps are as follows:
    a.制备四氧化三铁纳米微粒内核a. Preparation of ferroferric oxide nanoparticle core
    配制二价铁盐溶液，加至含有柠檬酸钠和硝酸盐的碱性溶液中，在25～90℃条件下搅拌5分钟以上，得到的沉淀离心分离后，用碱液洗涤三次，得到稳定的磁悬浮液；The divalent iron salt solution is prepared, added to an alkaline solution containing sodium citrate and nitrate, and stirred at 25 to 90 ° C for 5 minutes or more, and the obtained precipitate is centrifuged and washed three times with an alkali solution to obtain a stable Magnetic suspension
    b.制备表面硅包覆的纳米磁微粒b. Preparation of surface silicon coated nano magnetic particles
    用步骤a制备的纳米磁微粒，磁性分离，调节pH值为10～13，将正硅酸乙酯溶解于乙醇中，并按一定比例加入至分散液中，30～50℃条件下混合物搅拌4～5小时，在磁微粒表面生成的一层二氧化硅得到的包覆；磁分离得到表 面硅包覆的磁微粒；The nano magnetic particles prepared by the step a are magnetically separated, adjusted to a pH of 10 to 13, and the ethyl orthosilicate is dissolved in the ethanol and added to the dispersion in a certain ratio, and the mixture is stirred at 30 to 50 ° C. ~5 hours, a layer of silicon dioxide formed on the surface of the magnetic particles; magnetic separation to obtain a table Surface-coated silicon-coated magnetic particles;
    c.制备表面羧基修饰的磁微粒c. Preparation of surface carboxyl modified magnetic particles
    按酸酐基团与氨基摩尔比(1.2～2)∶1将称取氨基化硅烷与酸酐在无水溶剂中搅拌10到60分钟，得到羧基化硅烷。将羧基化硅烷溶解于无水乙醇中，加入1.2制备的纳米磁微粒，用碱液调节pH值至10～13，30～50℃搅拌4～5小时，然后用无水乙醇与纯化水洗涤，磁分离，得到表面羧基修饰的纳米磁微粒；The aminated silane and the acid anhydride are stirred in an anhydrous solvent for 10 to 60 minutes in a molar ratio of the acid anhydride group to the amino group (1.2 to 2): 1, to obtain a carboxylated silane. The carboxylated silane is dissolved in absolute ethanol, and the nano magnetic particles prepared in 1.2 are added, and the pH is adjusted to 10 to 13 with an alkali solution, stirred at 30 to 50 ° C for 4 to 5 hours, and then washed with absolute ethanol and purified water. Magnetic separation to obtain nano-magnetic particles modified with surface carboxyl groups;
    d.制备免疫磁微粒d. Preparation of immunomagnetic particles
    将步骤c制备的羧基磁微粒用pH值为6.0～7.0的MES缓冲液洗涤三次，再分散于此缓冲液中，用EDC与Sulfo-NHS在室温下活化振荡反应0.5～1小时后，磁分离，洗涤除去未反应的试剂，加入抗原、抗体或其它生物活性分子，室温振荡反应3h，磁分离，缓冲液洗涤后即得。The carboxyl magnetic particles prepared in the step c are washed three times with MES buffer having a pH of 6.0 to 7.0, and then dispersed in the buffer, and activated by shaking reaction with EDC and Sulfo-NHS at room temperature for 0.5 to 1 hour, and then magnetic separation. Washing to remove unreacted reagents, adding antigen, antibody or other biologically active molecules, shaking reaction at room temperature for 3 h, magnetic separation, and buffer washing.
  3. 如权利要求2所述的制备方法，其特征在于，所述氨基包括伯胺基和仲胺基。The method according to claim 2, wherein the amino group comprises a primary amino group and a secondary amino group.
  4. 如权利要求3所述的制备方法，其特征在于，所述二价铁盐为硫酸亚铁、氯化亚铁中的一种；硝酸盐为硝酸钠、硝酸钾中的一种；碱液为氨水、四甲基氢氧化铵、氢氧化钠中的一种。The preparation method according to claim 3, wherein the divalent iron salt is one of ferrous sulfate and ferrous chloride; the nitrate is one of sodium nitrate and potassium nitrate; and the alkali solution is One of ammonia water, tetramethylammonium hydroxide, and sodium hydroxide.
  5. 如权利要求4所述的制备方法，其特征在于，所述二价铁盐摩尔浓度比为0.01mol/L～1mol/L；柠檬酸钠浓度为0.5mmol/L～10mmol/L，硝酸盐浓度为0.2mol/L～3mol/L。The preparation method according to claim 4, wherein the molar ratio of the divalent iron salt is from 0.01 mol/L to 1 mol/L; the concentration of sodium citrate is from 0.5 mmol/L to 10 mmol/L, and the nitrate concentration is It is 0.2 mol/L to 3 mol/L.
  6. [根据细则91更正 20.07.2017]　
    如权利要求5所述的制备方法，其特征在于，所述正硅酸乙酯与四氧化三铁的质量比为(1～5)∶(1～20)。     [Correct according to Rule 91 20.07.2017]
    The method according to claim 5, wherein the mass ratio of the tetraethyl orthosilicate to the ferric oxide is (1 to 5): (1 to 20).
  7. [根据细则91更正 20.07.2017]　
    如权利要求6所述的制备方法，其特征在于，所述酸酐为丁二酸酐、戊二酸酐、己二酸酐、马来酸酐、邻苯二甲酸酐中的一种；氨基化硅烷为γ-氨丙基三乙氧基硅烷、γ-氨丙基三甲氧基硅烷、二乙烯三氨基丙基三甲氧基硅烷、N-(β-氨乙基)-γ-氨丙基三乙氧基硅烷、N-(β-氨乙基)-γ-氨丙基甲基二甲氧基硅烷中的一种；无水溶剂为N，N-二甲基甲酰胺、二甲亚砜中的一种。     [Correct according to Rule 91 20.07.2017]
    The preparation method according to claim 6, wherein the acid anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, maleic anhydride, and phthalic anhydride; and the aminated silane is γ- Aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, diethylenetriaminopropyltrimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane One of N-(β-aminoethyl)-γ-aminopropylmethyldimethoxysilane; the anhydrous solvent is one of N,N-dimethylformamide and dimethyl sulfoxide .
  8. [根据细则91更正 20.07.2017]　
    如权利要求9所述的制备方法，其特征在于，所述酸酐与氨基化硅烷的摩尔比为(1～5)∶(1～20)。     [Correct according to Rule 91 20.07.2017]
    The process according to claim 9, wherein the molar ratio of the acid anhydride to the aminated silane is (1 to 5): (1 to 20).
  9. [根据细则91更正 20.07.2017]　
    如权利要求8所述的制备方法，其特征在于，所述磁纳米微粒与EDC，Sulfo-NHS的质量摩尔比为1g∶(1～100mmol)∶(1～50mmol)；加入的所述磁微粒与抗体的质量比1g∶(0.1～10mg)。     [Correct according to Rule 91 20.07.2017]
    The method according to claim 8, wherein the mass ratio of the magnetic nanoparticles to EDC and Sulfo-NHS is 1 g: (1 to 100 mmol): (1 to 50 mmol); and the magnetic particles are added. The mass ratio to the antibody is 1 g: (0.1 to 10 mg).
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