Immunomagnetic microspheres (IMB) are a new type of material developed by combining immunology and magnetic carrier technology. IMB is a magnetic microsphere coated with monoclonal antibody, which can specifically combine with the target substance containing the corresponding antigen to form a new complex.
When passed through a magnetic field, this complex can be retained and separated from other components, a process known as immunomagnetic separation (Immunomagntic Separation). Immunomagnetic separation is simple and easy, with high separation purity, retains the activity of the target substance, and is efficient, fast, and low-toxic. It can be widely used in cell separation and purification, immune detection, nucleic acid analysis and genetic engineering, as a carrier for targeted drug release, etc. field
Magnetic microspheres are composed of carrier microspheres and ligands. Ideal magnetic microspheres are uniform spherical particles with superparamagnetic and protective shells.
1. Performance introduction of magnetic microspheres
1. Magnetic material
γ-Fe2O4, Me-Fe2O4 (Me = Co, Mn, Ni), Fe3O4, Ni, Co, Fe, Fe-Co and Ni-Fe alloys, etc. Currently, iron and its oxides are the most studied and widely used (Fe, Fe2O4 and Fe3O4, etc.).
2. Polymer materials
Polyethyleneimine, polyvinyl alcohol, polysaccharides (cellulose, agarose, dextran, chitosan, etc.) and bovine serum albumin, etc. The surface often has chemical functional groups, such as -OH, -NH2, -COOH and -CONO2, etc., so that the magnetic microcarriers can be coupled to almost any biologically active protein.
3. Functional base
The ligand must have the characteristics of biological specificity, and the combination of the carrier and the microsphere and the ligand will not affect or change the original biological characteristics of the ligand, so as to ensure the special recognition function of the microsphere.
Magnetic polymer microspheres determine the size and shape of immunomagnetic microspheres. Hirschein obtained the relationship between the applied magnetic field force and the magnetic microspheres as:
F=(Xv - Xv0) VH (dH/dX)
Where F is the force of the external magnetic field; Xv is the magnetic susceptibility of the magnetic microsphere; Xv0 is the magnetic susceptibility of the medium; H is the external magnetic field; V is the volume of the magnetic microsphere; dH/dX is the magnetic field strength. The force F experienced by magnetic particles in a magnetic field is proportional to the size of the particles. When the particle diameter D>10μm, it can be separated under a weak magnetic field, easy to precipitate, and the amount of adsorbed biomolecules is also small; when the diameter D<0.03μm, the particles can be stably dispersed in the solution, and the separation requires a large magnetic field strength. The selected particle size range should be determined according to the characteristics of the separation system.
F is also related to the magnetic susceptibility of magnetic microspheres. The magnetic susceptibility of microspheres is directly determined by the composition and size of the magnetic powder as the magnetic core. The commonly used oxide-deficient, when the crystal of its structure is less than 30nm, becomes a superparamagnetic material. When the crystal is larger than 30nm, it becomes ferromagnetic. Large specific surface and high dispersion stability: With the refinement of microspheres, when the particle size reaches nanoscale, the specific surface increases sharply, the density of functional groups on the surface of microspheres and the selective adsorption capacity become larger, and the time to reach adsorption equilibrium is greatly shortened , the dispersion stability of the particles is also greatly improved.
4. Soft magnetic effect
Under the action of an external magnetic field, the soft magnetic polymer microspheres can generate magnetism and move in a directional manner, and the magnetism disappears after the magnetic field is removed, so that separation and magnetic guidance can be conveniently performed.
5. Biocompatibility
Nanomagnetic microspheres have good biocompatibility with most biopolymers such as polysaccharides and proteins. In bioengineering, especially in the field of biomedicine, it is very important to have good biocompatibility.
6. Function-based characteristics
The functional groups on the surface of magnetic microspheres can be covalently linked to various active groups of biopolymers such as -OH, -COOH, -NH2, and can stably immobilize biologically active substances (such as antibodies, antigens, receptors, etc.) bodies, enzymes, nucleic acids and drugs, etc.).
Due to the above characteristics of nano-magnetic polymer microspheres, according to different needs, through copolymerization and surface modification, a variety of specific reactive functional groups can be endowed on the surface, and then combined with various functional substances, it is widely used in organic synthesis. And chromatographic fillers, cell labeling and separation, immobilized enzymes and bacteria, nucleic acid separation and purification, biochip materials, industrial wastewater purification, carriers for targeted drug delivery systems, and immunoassays, etc.
2. Preparation of immunomagnetic microspheres
1. Basic technical route
Make microspheres of magnetic materials, and then introduce active groups on the surface of the microspheres, and bind the antibody to the carrier through the surface coupling reaction of the carrier to form immunomagnetic microspheres.
2. Performance of high-quality microcarriers
Appropriate and uniform magnetic response intensity, small and uniform particle size, stable and uniform, specific adsorption surface properties.
3. Preparation of magnetic microcarriers
(1) Embedding method
The magnetic particles are dispersed in the polymer solution, and the magnetic polymer microspheres are obtained by atomization, flocculation, deposition, evaporation and other means.
(2) Monomer polymerization method
In the presence of magnetic particles and monomers, add initiators, stabilizers, etc. to polymerize core/shell magnetic polymer microspheres. Combination of antibody and magnetic carrier: After the polymer layer on the surface of the magnetic microcarrier is activated, it is suspended in the antibody solution and shaken for a period of time at room temperature or low temperature (ice-water bath) to connect the antibody to the surface of the microsphere and obtain an immunomagnetic microsphere. ball.
3. Application of Immunomagnetic Microspheres
1. For cell separation and purification
There are two ways to use IMB to separate cells; the method of directly separating target cells from the cell mixture is called positive separation; the method of using immunomagnetic beads to remove irrelevant cells so that target cells can be purified is called negative separation. Immunomagnetic bead technology can be used to separate various human cells such as erythrocytes, peripheral blood eosinophils/basophils, neural stem cells, hematopoietic cells, T lymphocytes, γδT lymphocytes, human joint synoviocytes, dendritic cells, endothelial cells cells, and various tumor cells.
2. In vitro cell expansion
Dendritic cells (Dendritic cells, DCs), hematopoietic stem cells, progenitor cells and other cells have great application value in scientific research and clinical practice, but their content in the body is small and widely distributed, and it is difficult to obtain a large number of high-purity cells, which limits the field development. In vitro amplification supplemented by immunomagnetic bead technology is expected to solve this problem. In this process, the cells to be expanded are separated and purified with immunomagnetic microspheres, and cultured with a specific combination of factors. Many researchers use this method to find the best combination of cytokines for expansion and the best time for transplantation.
3. Immunoassay
Immunomagnetic microspheres can simply and quickly enrich and remove cancer cells from blood or bone marrow, and are widely used in disease detection, cancer treatment and autologous bone marrow transplantation, and are also used to separate fetal cells from maternal peripheral blood for non-invasive Prenatal diagnosis.
The use of immunomagnetic bead separation technology in microbial detection can accurately and quickly detect Coli O 157 in samples, which is of great significance for food hygiene and prevention of disease transmission. The combination of PCR technology and immunomagnetic bead technology plays a very important role in molecular biology and medical diagnostics. The application of this research in medical testing can easily and quickly diagnose bladder cancer, breast cancer, prostate cancer, and peritoneal cancer. Gastric cancer, epithelial tumor cells, etc., make the application of immunomagnetic separation technology more extensive.
4. Application in nucleic acid and genetic engineering
Immunomagnetic spheres can be regarded as miniature ligands in affinity chromatography technology. With the help of avidin-biotin (Biotin-Avidin) system, immunomagnetic spheres can bind to non-proteins. There is a high degree of interaction between biotin and avidin. The affinity of the two, the combination of the two is rapid, specific and stable, and the application in molecular biology, medicine, immunohistochemistry and other fields is becoming more and more extensive. After combining with the biomagnetic bead technology, it has produced an attractive Development prospects, and widely used in the separation and purification of RNA, mRNA, nucleic acid fragments, etc. and related research.
5. For typing
The immunomagnetic bead method can be applied to the rapid matching of donors and recipients for clinical organ transplantation. Under high gradient magnetic field, T and B lymphocytes in venous or peritoneal blood were separated by immunomagnetic bead method, and the separated lymphocytes were used for HLA-class II antigen typing. For example, using magnetic bead technology and monoclonal antibody reagents to establish a new method that can complete HLA class I and II antigen typing in 1.5 hours, immunomagnetic bead separation technology can also be used for HLA typing of kidney transplant donors and recipients, and to explore blood Correlation between the therapeutic effect of repeated platelet transfusions and HLA in patients with this disease.
6. Used as a carrier for targeted drug delivery system
Immunomagnetic microspheres as the carrier of the targeted drug release system can make the anticancer drugs on the immunomagnetic microspheres more easily contact with cancer cells. Guided to a specific target area in the body, it improves the effect of killing cancer cells. Many researchers have used different methods to make immunomagnetic microspheres targeting different cancer cells as the carrier of the targeted drug delivery system, and the experiments have confirmed that the drug delivery carrier has good efficacy.
