IVD research dog, would you choose luminescent magnetic beads?
Known for its high sensitivity, chemiluminescent immunoassays (CLIAs) can detect analytes at low concentrations and provide good detection limits over a wide dynamic range. Immunomagnetic beads (IMB) have the advantages of simple operation, high separation efficiency, large specific surface area and good physical stability. They are a kind of magnetic separation carrier with excellent performance and a general tool for purification, detection and quantitative analysis of complex analytes. As a solid-phase carrier, magnetic beads are combined with CLIA. Under the action of an external magnetic field, they can be quickly separated from the substrate liquid phase. Magnetic beads immobilize antigens on the magnetic beads in the immunoassay reaction due to their high-efficiency separation and enrichment. The effect of antibodies was significantly reduced.
Advantages of magnetic beads as carriers for immune response and signal collection:
1) Magnetic beads have a large specific surface area and can bind more protein molecules, which can improve the detection range.
2) The chemical groups on the surface of the magnetic beads form a covalent coupling with the protein, which is stronger and more stable than physical adsorption.
3) The magnetic beads are evenly suspended in the reaction solution, which greatly increases the contact area with the analyte in the sample, reduces the sample volume required for the reaction, and at the same time reaches the dynamic equilibrium of the reaction faster, speeds up the reaction speed, and saves reaction time.
4) The combination of magnetic beads connected with multiple capture proteins and multi-labeling technology can realize the simultaneous detection of multiple analytes in the same sample, and realize automatic individualized detection.
1. Types and structures of magnetic beads
1.1 Common types of magnetic beads
1) Carboxyl Magnetic Beads (Carboxyl Mag)
Carboxyl carboxyl magnetic beads are high-performance magnetic beads for chemiluminescent reagents. Carboxyl functional groups are used for protein or nucleic acid coupling. They can play outstanding performance in enzyme immunoassay, immunoprecipitation, and Western blot analysis.
2) Tosyl Magnetic Beads (Tosyl Mag)
Because the magnetic bead coating contains toluenesulfonyl groups, molecules such as antibodies containing amino groups can be directly immobilized on the surface of magnetic beads by chemical binding without the need for carboxylating agents.
3) Streptavidin Magnetic Beads (Streptavidin Mag)
Magnetic beads coupled with streptavidin can specifically absorb biotin-labeled molecules with high purity, and the hydrophilic polymer coating on the surface of magnetic beads will not affect enzymatic reactions and PCR nucleic acid amplification.
1.2 Structure
The magnetic bead structure is generally composed of a magnetic core, a polymer coating and a functional base layer. Under the action of an external magnetic field, the magnetic beads make directional movement in the magnetic field, supplemented by micro-control and detection means, which can realize the magnetic bead positioning and medium separation.
1) Core: It is mainly composed of magnetic materials such as pure metals (such as cobalt, nickel and iron) or their oxides. In addition, magnetic materials such as CoPt3, FeCo and FePt can also be used.
2) Polymer coating: The material is generally polystyrene and polyvinyl chloride, which is mainly used to stabilize the surface of the newly formed magnetic beads and prevent the aggregation of magnetic beads. The polymer coating can be combined with a variety of active substances, such as antigens, antibodies, nucleic acids, etc.
3) Functional base
The stability of the surface of protein or antibody-conjugated magnetic beads mainly depends on the surface properties. When the surface of the magnetic bead is not modified, the antibody is passively adsorbed to the surface of the magnetic bead through hydrophobic interaction. This approach cannot control the final orientation of the attached molecules, nor can it guarantee the specificity and stability of the capture molecules. To ensure coupling stability and capture specificity, chemically modified surfaces and biologically modified surfaces can be selected.
u Chemically modified surface:
①Classical chemically modified surface:
Antigens or antibodies are directly and passively adsorbed on the surface of chemically modified magnetic beads. The active group utilizes the surface area of the magnetic beads to provide a large number of sites for the antigen or antibody to be covalently attached to the magnetic beads. Classic groups added to surfaces are carboxyl, amino, hydroxyl and sulfate.
Table 1. Typical chemical modification and activation methods of magnetic beads
Chemical modification and activation of magnetic beads:
carboxyl modification
Add carbodiimide (EDC), N-hydroxysuccinimide (NHS) or sulfo-NHS and ethyl (dimethylaminopropyl) for activation.
Amino modification
Activate protein surface carboxyl groups.
Hydroxyl modification
Activate in a non-aqueous solution to avoid hydrolysis of the intermediate.
② Pre-activated modified surface:
There is no need for initial activation before molecules are attached to the surface preactivated modified magnetic beads, and stable covalent coupling can be formed under appropriate buffer, pH and temperature conditions.
Table 2. Preactivation Modifications and Binding Groups of Magnetic Beads
Magnetic bead preactivation modification and binding groups:
Tosyl modification
Neutral pH value: bind protein sulfhydryl; alkaline pH value: bind amino group.
epoxy group modification
Slightly alkaline pH: binding to thiol groups; higher pH: binding to amino groups; strongly alkaline pH: binding to hydroxyl-containing ligands.
Chloromethyl modification
Neutral pH at room temperature: bound to amino groups.
ubiologically modified surface (surface biofunctionalization):
Biofunctionalized magnetic beads are widely used to capture specific molecules or cells due to their superparamagnetism. Biological modifications differ from chemical modifications in that they link molecules non-covalently. In the field of biotechnology, biofunctionalized magnetic beads are usually used in separation processes such as protein/molecule purification, and as solid phase carriers in in vitro diagnostic reagents.
①Protein A or G binds certain immunoglobulin subtypes with very high affinity. Protein A binds to the Fc region of most Ig, and protein G binds to the Fc or Fab region of Ig, which can be used to immobilize antibodies;
②Streptavidin has a very high affinity, and the combination between streptavidin and biotin can withstand extreme conditions such as high temperature and a wide range of pH values.
Chemical modification: Magnetic beads are covalently coupled to antibodies through chemical modification groups such as -COOH, -NH2, -OH, -SH on the surface
Advantages: simple surface modification, stable coupling
Disadvantages: prone to aggregation and non-specific binding
Biological modification: Magnetic beads are non-covalently coupled to antibodies through biological modifications such as surface streptavidin, anti-IgG, protein A or G
Advantages: biomagnetic separation without additional washing; can withstand extreme conditions such as high temperature
Cons: Expensive
2. The principle and application of immunomagnetic beads
2.1 Principle
Magnetic beads generally have superparamagnetism, which can realize rapid separation of bound and unbound proteins under the action of a magnetic field, simplify operation and shorten reaction time. The surface of magnetic beads binds active proteins through externally modified functional groups, and acts as a carrier for antigen-antibody reactions. The mode of action of magnetic beads is divided into direct action and indirect action. The direct action is to use antibody/antigen-coated magnetic beads to directly bind with specific antigen/antibody substances to form magnetic bead-immune complexes. The indirect effect is to coat the magnetic beads with the secondary antibody, incubate the antigen with the primary antibody, and then add the magnetic beads coupled with the secondary antibody to form a magnetic bead-secondary antibody-primary antibody-antigen complex. The complex has strong magnetic responsiveness, and moves directionally under the action of magnetic force, so that the complex can be separated from other substances in the liquid to achieve the purpose of separating, concentrating and purifying specific proteins.
2.2 Application
Immunomagnetic beads immobilized antigen or antibody have been widely used in immunoassay, cell separation, purification of biological macromolecules, molecular biology and so on. Immunomagnetic beads can also be used in clinical targeted drug delivery, magnetic resonance imaging, and intercellular hyperthermia for targeted destruction of tumors.
1) Immunoassay
In immunoassays, immunomagnetic beads are used as solid-phase carriers. Antibodies and antigens are specifically combined on the magnetic beads to form antigen-antibody complexes. Under the action of magnetic force, specific antigens are separated from other substances. This sub-magnetic separation has the advantages of high sensitivity, fast detection speed, high specificity and good repeatability. Magnetic beads can also detect surface molecules and determine cell types through polarity phenomena, and can be used for the detection of hormones, neurotransmitters, cytokines, tumor-associated antigens and other substances.
2) Cell isolation
Using magnetic beads and antibodies, cells can be separated under the action of a magnetic field, which is widely used in:
① Detection of a small amount of tumor cells in body fluids to improve the early diagnosis rate of tumors;
② Rapid and efficient separation of T and B cells for HLA tissue typing in organ transplantation;
③ Pretreatment of bone marrow transplantation to improve the success rate of transplantation;
④Separation or removal of specific cell components for various medical and scientific research projects.
3) Biological purification
Magnetic beads can be used as miniature ligand carriers to immobilize antigens or antibodies on the matrix to form specific adsorption, and then perform magnetic affinity extraction without centrifugation and filtration. It can be used to separate and purify macromolecules, DNA, RNA, DNA binding proteins, mRNA and other substances.
4) Molecular biology applications
Magnetic beads can be combined with non-proteins with the help of avidin-biotin system, and the single-strand separation method of magnetic beads solid phase can directly separate the PCR double-strand product or sequence its single-strand.
