At present, the application of magnetic microspheres in biomedicine is represented by immunomagnetic microspheres, which are developing rapidly. There are already products on the market, and there are many successful applications. Magnetic nanomaterials are an important part of magnetic microspheres, so the application of magnetic microspheres is also the application of magnetic nanomaterials.
Preparation and properties of magnetic nanomaterials
Commonly used magnetic materials are ferric oxide, ferric oxide, iron-cobalt alloy, etc. These magnetic materials have good magnetic responsiveness, and these magnetic materials whose size is in the nanometer scale can be obtained conveniently by adopting an appropriate method. For example: Dissolve a certain amount of magnetic material in an appropriate amount of distilled water, filter and mix, add a certain amount of distilled water to dilute, stir evenly, and add an appropriate surfactant as a dispersant. Keep stirring at a certain temperature, add the solution to the system at a certain speed, and continue stirring for half an hour after the dropwise addition. Take it out, place it on a magnet, make the iron oxide particles settle, remove the supernatant, then add an appropriate amount of aqueous solution of a dispersant, disperse with ultrasonic waves, and filter to obtain a color colloid solution of iron oxide magnetic nanoparticles.
The concentration of life in the preparation process has a great influence on the performance of iron oxide nanoparticles. If the concentration of lye is low, the magnetization of the magnetic material will be low; the dropping speed of lye affects the performance of the magnetic material. The smaller the particle size, the lower the magnetization; the reaction temperature of the system also affects the temperature, the larger the particle size and the stronger the magnetization.
The magnetic nano-materials obtained by the above method can be properly diluted with water, and electron microscope photos can be taken, and the size and distribution of the particle size can be measured by an image analyzer, or directly measured by a laser particle size analyzer, and nano-materials of different sizes can be obtained. Magnetic materials are available with an average particle size of a few nanometers, generally with a normal distribution. X-ray diffractometer can also be used to analyze the structure of magnetic nanomaterials, and magnetometer can be used to measure its magnetization.
Preparation of magnetic microspheres with magnetic nanomaterials
Magnetic microspheres can be prepared from magnetic nanoparticles and polymer framework materials. The polymer materials include polystyrene, silane, polyene, polyacrylic acid, starch, dextran, gelatin, albumin, ethyl cellulose, etc. There are natural and synthetic ones, which can be used alone or in combination as a skeleton material. These skeleton materials should be stable in nature, high in strength, and have no toxic side effects.
The method of preparing magnetic microspheres can be divided into one-step method and two-step method: the one-step method is to add magnetic nanomaterials before forming the balls, and the polymer wraps them in the balls; the two-step method is to prepare non-magnetic beads first, and then the magnetic material enters it through processing, and finally the magnetic nanoparticles exist in the bone plus material of the microsphere in a dispersed form.
The one-step method was developed earlier, and there are many methods, only the following four are introduced.
1. Disperse magnetic nanomaterials (such as ferric oxide nanoparticles, etc.) in water, add monomers of high molecular polymers, and then add initiators to initiate polymerization under appropriate conditions, so that the monomers in ferric oxide The surrounding nanoparticles aggregate to form magnetic microspheres. Magnetic microspheres with synthetic polymer materials as the skeleton are mostly prepared by this method.
2. Disperse the magnetic nanomaterials in the aqueous solution of the polymer framework material, add an appropriate surfactant, emulsify in a hydrophobic solvent to form a W/O emulsion, and use the thermal curing method or cross-linking curing method to make the polymer The framework material solidifies into magnetic microspheres. Magnetic microspheres based on natural polymer materials are mostly prepared by this method.
3. Precipitate Fe2+ and Fe3+ in an alkaline solution to make superparamagnetic iron oxide, and then coat it with silane to form microspheres. The prepared silane magnetic microspheres can be dispersed in an aqueous medium without rapid precipitation, and can be conveniently recovered by a magnetic field.
4. Using the magnetite itself as part of the oxidation-reduction system, the polymer can completely wrap around the magnetite. Polymerization is initiated by iron ions diffusing out of the magnetite particles and becoming free radicals through the reduction of persulfate. With this method, a kind of aqueous gel magnetic microsphere containing acrylic resin can be prepared. There are few literatures on the two-step method.
Preparation, performance and action principle of immunomagnetic microspheres
1. Preparation of immunomagnetic microspheres Immunomagnetic Microspheres (IMMS), or Immunomagnetic Beads (IMB), are magnetic microspheres with monoclonal antibodies bound to their surfaces. Due to the need to bind appropriate antibodies on the surface of the magnetic microspheres, it is required that the magnetic microspheres used can bind to the monoclonal antibody through the chemical gene on its surface, or have a large surface adsorption force to be able to firmly bind to the monoclonal antibody. Cross-linked polystyrene microspheres have high strength and are easy to be chemically modified on the surface, so they are ideal skeleton materials for preparing immunomagnetic microspheres. There are two forms of connection between microspheres and antibodies: adsorption binding and covalent binding. Adsorption binding relies on the non-specific adsorption of the antibody on the surface of the microsphere, while covalent binding relies on the covalent reaction between the active groups on the surface of the microsphere and the antibody. The adsorption binding is only possible when the microsphere has a very large surface area. In this way, for the microsphere with a relatively flat surface, it is necessary to improve its antibody binding force by treating the surface to ensure that the surface of the IMMS has enough antibodies. The surface-treated magnetic microspheres are cultured with monoclonal antibodies in an appropriate buffer, and the antibodies quickly bind to the magnetic microspheres through physical adsorption. Reactions are covalently bound to the surface of magnetic microspheres.
2. The performance and principle of immunomagnetic microspheres Immunomagnetic microspheres are mainly used in cell separation and other aspects. Because it can specifically bind to the target substance and make it magnetically responsive, the immune magnetic microspheres are co-incubated with a complex mixture containing the target substance (substance to be separated). The immune microspheres can selectively bind to the target substance through the antigen-antibody reaction. When the compound passes through a magnetic field device, the target substance bound to the immunomagnetic microsphere will be retained by the magnetic field, thereby separating it from other complex substances. Immunomagnetic microspheres for cell separation have the following conditions: stable chemical properties, no aggregation; no non-specific binding to cells; strong binding between magnetic microspheres and antibodies; uniform size of magnetic microspheres and good magnetic responsiveness, the content of magnetic nanomaterials is uniform; the size of magnetic microspheres is appropriate, and it is not easy to be swallowed by cells. The combination of IMM and cells can adopt two methods, direct method and indirect method. The direct method refers to the fact that the antibody is attached to the magnetic microspheres one after another, and then binds to the target cells. The indirect method refers to mixing and culturing the cells with specific antibodies first, so that the specific antibodies bind to the cell surface, and then adding magnetic microspheres pre-treated with anti-mouse IgG (secondary antibody). The magnetic microspheres are indirectly bound to the target cells. The direct method can reduce washing and incubation steps, but it is rarely used for IgM monoclonal antibodies. Compared with the direct method, the indirect method
In addition to an appropriate broad range, a panel of monoclonal antibodies can be used and thus better cell clearance will be obtained. However, after multiple washing steps, the specificity will also be reduced.
The development of monoclonal antibodies against monocytes has made it possible to isolate cells with specific surface markers. Generally, there are 3 different methods, namely flow cytometry (PACS), allogeneic erythrocyte rosette technology with secondary antibodies attached to the surface, and Panning technology with passive adsorption of antibodies on polystyrene tissue culture plates. Each of these techniques has its own drawbacks. FACS is expensive and complicated, and is often troubled by the capacity, activity, and sterility of the sorted cells; the red blood cell rosette technology cannot handle a large number of cells. In addition, there is no mature and simple method to separate antibodies Coupling to the red blood cell membrane; Panning technology also has many limitations, it is difficult to scale up, the steps are cumbersome, and cannot be quantified, and the target cells are often mixed with non-specific antibodies adsorbed on other cells at the bottom of the culture plate. Relatively speaking, immunomagnetic microspheres have the advantages of easy operation, rapid and complete separation, and high cell purity. Especially in terms of easy and time-saving operation, other separation methods are difficult to compare.