Bio-magnetic beads are very familiar to every molecular researcher. Because of their high specific surface area, uniform dispersion, resuspension, faster magnetic responsiveness, and more active functional groups, they are widely used in nucleic acid preparation, immunology, etc. Analysis, cell sorting, protein purification and other fields. This article will introduce why do magnetic beads have these characteristics?
1. Biological magnetic beads and structure
Bio-magnetic beads are a kind of magnetic microspheres with nanometer particle size, which are formed by high molecular polymer and inorganic magnetic nanoparticles. According to its structure, it can be divided into three categories:
The first category: core-shell structure. The core is a magnetic nanoparticle, and the shell is a polymer or inorganic nanomaterial, such as silanol magnetic beads. The surface is a silica matrix, and the interior is wrapped with magnetic nanoparticles. The surface is modified with a large number of silanol groups (hydroxyl groups). It is mainly used for nucleic acid extraction.
The second category: sandwich structure. The inner core is a polymer microsphere, the second layer is a magnetic nanoparticle, and the third layer is a functional polymer coating. For example, the innermost core of the fragment screening magnetic beads is polystyrene microspheres, the second layer is wrapped with a magnetic substance - triiron tetroxide, and the outermost surface is a polymer material modified by carboxyl groups. Various surface modification functional groups, such as carboxyl group, amino group, N-hydroxysuccinimide, protein A/G and streptavidin, etc., different kinds of biological ligands are covalently coupled to the surface of magnetic beads, applied to biotin Capture, immunoassay, cell sorting, etc.
The third category: diffuse structure. Magnetic composite microspheres are formed by dispersing magnetic particles in a polymer matrix. Most of them are based on natural agarose, which has a high load due to its rich porous structure. Its surface is modified with hydroxyl, amino or carboxyl functional groups, which can be used for protein immobilization. The surface can also be modified with metal ions, GSH, Strep-Tactin, DEAE, protein A/G, etc., which are commonly used in protein separation and purification.
2. The characteristics of biological magnetic beads
Bio-magnetic beads have a wide range of applications, mainly due to the following characteristics:
High surface area ratio. When the particle size becomes smaller, the surface area, surface energy and surface binding energy of nanomaterials increase rapidly, and the percentage of surface atoms increases significantly. When the particle size is as small as nanometers, almost all the atoms that make up the nanocrystals are distributed on the surface. Due to the lack of adjacent atoms around the surface atoms; there are many dangling bonds, which are unsaturated, and are easily combined with other atoms to stabilize, showing high chemical activity.
Magnetic responsiveness. Magnetic responsiveness is the response of magnetic microspheres to an applied magnetic field. Using this feature, the magnetic microspheres can be rapidly separated and magnetically guided under the action of an external magnetic field. The content of the magnetic core in the magnetic microsphere is an important parameter reflecting the magnetic responsiveness of the microsphere. When the diameter of the metal oxide particle as the magnetic core is less than 30 nm, it has superparamagnetic properties, that is, it has strong magnetism in the magnetic field. When there is an external magnetic field, the magnetic microspheres will carry nucleic acid or other biological macromolecules to move in a rapid direction to achieve the purpose of separation and purification.
Function-based properties. The polymer material that coats the magnetic particles has a variety of reactive functional groups, such as -OH, -COOH, -CHO, -NH2, -SH, etc., which can be connected to biologically active substances. Beads have different properties and have broad application prospects in the fields of cell separation, targeted drugs, nucleic acid hybridization, etc.
Biocompatibility. An important aspect of the application of magnetic microspheres in bioengineering, especially biomedical engineering, is biocompatibility. Most biopolymers such as polysaccharides and proteins have good biocompatibility. They are safe, non-toxic and degradable in the human body, and do not generate immunity with human tissues and organs. This property is especially important in targeted drugs.
3. The application of biological magnetic beads
There are various types of biological magnetic beads. Due to their different structures, modified functional groups, and different coating processes, the surface charge, repulsion, and the number of hydrogen bonds will be different, and the adsorption capacity and function will also change. Therefore, different kinds of magnetic beads play their respective functions in different fields. The application directions of several common magnetic beads are listed below:
Nucleic acid preparation
Magnetic beads for nucleic acid extraction: The silanol or carboxyl groups on the surface of the magnetic beads can specifically bind to nucleic acids in solution through hydrogen bonding and electrostatic interactions, so nucleic acids can be directly and rapidly separated from complex biological systems. At present, this technology is relatively mature, and it is widely used in the automatic extraction of nucleic acid, which greatly improves the throughput of nucleic acid extraction, and is simple and convenient. Jifan also provides customers with FineMag series products based on magnetic bead nucleic acid extraction, which are used for nucleic acid extraction of various sample types such as viral DNA/RNA, plasma free DNA, bacteria, plants, blood, tissue, blood slices, etc. Users can choose between pre-packed magnetic bead extraction reagents and non-pre-packed magnetic bead extraction kits according to their needs.
Fragment screening magnetic beads: Mainly used for NGS library construction, these magnetic beads use solid-phase reversible immobilization technology (SPRI) for nucleic acid purification and screening of specific fragments. By changing the buffer concentration of magnetic beads, nucleic acid fragments with larger molecular weights can be preferentially adsorbed, and fragments of different molecular weights can be recovered, eliminating the inconvenience caused by gel cutting and purification.
Hybrid capture magnetic beads: streptavidin and biotin form a complex, which is the strongest known combination of non-covalent interactions between proteins and other molecules. This complex is not affected by pH, temperature, organic Extreme factors such as solvents. Streptavidin magnetic beads can be combined with biotin-labeled high-purity antibody polypeptide molecules or nucleic acid molecules to further separate hybrid complexes. This method is widely used in the process of NGS library construction.
Magnetic beads are mainly used in clinical in vitro diagnostic (IVD) detection technology in immunoassays. By connecting specific antibodies with magnetic beads to find their acting protein antigens, separate and purify them, and finally detect the number of protein antigens. The main techniques used include chemiluminescence immunoassay (CLIA), microfluidic magnetic sensitivity immunoassay (MIA), Fluorescence immunoassay (IFA), etc., are widely used in real-time detection of POCT.
Magnetic particle chemiluminescence immunoassay technology is an emerging analytical method that combines magnetic separation technology, chemiluminescence technology and immunoassay technology. Sensitivity, as well as the specificity of immunoassays, play an irreplaceable role in the field of bioanalysis. For example, the acridine ester chemiluminescence assay can be used to measure free thyroxine (FT4), creatine kinase isoenzyme (CKMB), N-terminal precursor B-type natriuretic peptide (NT-proBNP), total prostate specific antigen (TPSA), free prostate specific antigen (FPSA) and other immunoassays. Magnetic particle chemiluminescence immunoassay (double antibody sandwich) can be used to detect myoglobin (MYO).
The surface of the magnetic beads is modified with different functional groups for the immobilization of different kinds of biological ligands. Such as metal ions, GSH, Strep-Tactin, DEAE, protein A/G, etc., for protein separation and purification. At present, the commonly used magnetic beads for histidine-tagged protein purification, DEAE protein purification magnetic beads, heparin magnetic beads, GST-tagged protein purification magnetic beads, Flag-tagged protein purification magnetic beads, antibody purification magnetic beads (proteinA/Protein G/Protein L ), Strep-tag II protein purification magnetic beads, etc. The application of antibody (protein)-coated immunomagnetic bead purification technology does not require complex chromatography equipment, and there is no limit to the clarity of the sample. It only needs a simple magnetic adsorption step to carry out the separation work easily, effectively solving the traditional chromatography. inadequacies of technology.
Magnetic bead cell sorting (MACS) is based on the combination of cell surface antigens and specific monoclonal antibodies attached to magnetic beads, and the cells labeled with magnetic beads are separated from other cells under the action of an external magnetic field, so as to realize the enrichment of target cells. collection and purification. The existing magnetic bead cell sorting methods mainly include positive sorting (positive selection): the cells bound by magnetic beads are the cells to be separated, which are suitable for flow analysis and cell-based analysis. Negative sorting (negative sorting): The magnetic beads bind the unwanted cells, and the cells free in the supernatant are the desired cells. Multiple sorting strategies are also possible: first "negative sorting" and then "positive sorting". When using nanoscale magnetic beads for cell sorting, the size of the magnetic beads and its composition make it biodegradable without activating cells or affecting the function and vitality of cells, and the physiological functions of cells remain unchanged. It has a wide range of applications in tumor research, cellular immunotherapy, and single-cell analysis.
Magnetic transfection technology is a novel, simple and efficient method for transfecting cells. This technology combines magnetic particles and certain biological macromolecules through chemical covalent bonds or physical adhesion to form magnetically responsive microparticles. Then, it is coupled with traditional viral or non-viral vectors through the surface groups of the magnetic particles, and then combined with the target gene to form gene-loaded magnetic microspheres. Under the action of an external gradient magnetic field, the magnetic microspheres will focus on the target organ or tissue with the guidance of the magnetic field force. Under the endocytosis of cells, the magnetic microspheres enter the cells and the target genes are released. The co-modification strategy of cationic polymer and biocompatible polymer on the surface of the nano-magnetic transfection carrier greatly increases the dispersibility, colloidal stability and biocompatibility of the gene-loaded magnetic transfection carrier. delivery research.
Magnetic hyperthermia refers to injecting superparatic oxide magnetic beads into the tumor lesion area, and applying an alternating magnetic field to heat the lesion area to a specific temperature due to the magnetothermal effect, thereby killing tumor cells. This technology has the characteristics of targeting, less toxic and side effects, and easy to combine with other methods. At present, Germany and the United States have entered the clinical research stage, and some patients with breast cancer, prostate cancer and other tumors have been cured, showing good development prospects. The products developed by MagForce in Germany based on the nano-iron oxide magnetic liquid (Nanotherm®) glioma magnetic induction hyperthermia technology have been commercialized in Europe.
Another important application in tumor therapy is drug-targeted therapy. Magnetic drug preparation is a magnetic targeted drug delivery system made by encapsulating drugs and magnetic substances together in a polymer carrier. An external magnetic field guides the directional movement of the drug and is positioned and concentrated in the target area, and the drug is released in the magnetic field. The surface-modified organic polymers or inorganic metals or oxides of the magnetic nanobeads make them biocompatible so that the drug delivery to specific sites can eliminate the side effects of the drugs and reduce the drug dosage. In my country, the magnetic guidance carrier doxorubicin interventional therapy (MTC-DOX) has been applied to the clinical research of primary liver cancer.
In vivo diagnosis
Magnetic nanoparticles are mainly used in MRI for in vivo diagnosis. Due to the development of MRI in diagnosis, a new class of drugs, magnetic drugs, has emerged. These drugs are administered to patients to increase the contrast (contrast) of normal and diseased tissue and/or show organ function or blood flow. MRI contrast agents mainly include gadolinium and superparamagnetic nano-iron tetroxide particle contrast agents. Compared with gadolinium agents, ferric oxide nanoparticles are widely used in magnetic resonance imaging studies due to their high sensitivity, biocompatibility, and superparamagnetic properties. The magnetic resonance contrast agent products developed by Advanced Magnetic Company of the United States and Schering Company of Germany have been approved by the FDA.
As an interdisciplinary product, bio-magnetic beads have been gradually applied in the fields of bioengineering, cytology and biomedicine. High-sensitivity, high-throughput, and high-efficiency magnetic separation and purification technology have shown broad application prospects in the clinical diagnostic market and biomedicine. With the continuous development of bio-magnetic bead technology, there will be more application directions and huge market demand.
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