Immunoassay is one of the most important methods in the fields of clinical, pharmaceutical analysis, toxicological analysis and environmental analysis due to its high sensitivity, high specific affinity, rapid detection and possible analysis of difficult matrices without extensive pre-treatment. The magnetic bead-based immunoassay is a novel type of immunoassay technology that has emerged after radioimmunoassay, enzyme-linked immunoassay, fluorescence immunoassay, colloidal gold immunoassay, chemiluminescence immunoassay, etc.
Antibodies can specifically recognize and bind to the corresponding antigens, and this binding phenomenon can also occur in vitro. When antibodies and antigens are combined in vitro, there may be reactions (discoloration or precipitation) that can be seen by naked eyes, or reactions (fluorescence, resistance change) that can be detected with the aid of instruments. During the experiment, the known antibodies can be used to test the presence of the corresponding antigens in the specimen, and the known antigens can also be used to detect the presence of the corresponding antibodies in the specimen.
Why Choose magnetic beads (MBs) for Immunoassay?
The popularity of magnetic beads in the field of bioanalysis, especially in immunoassay, is mainly attributed to the following reasons.
- Huge surface area allows them to be utilized in immobilization of biomolecules such as enzymes, DNA, and antibodies.
- Chemical and physical stability, low toxicity, and high biocompatibility make them suitable for the immobilization of biomolecules.
- Efficient dispersing ability enablethem to shorten the reaction time between dissolved species and biomolecules.
- The preparation and separation of the sample are very simple and do notrequire pre-concentration.
- The miniaturization of the assay system can also be achievedby using microbeads as the mobile solid phase.
- Magnetic beads can be accumulated by using an external magnetic field, which can further promote fabrication of simple, rapid, sensitive, and user-friendly immunoassay systems.
Magnetic Bead-based Immunoassay Techniques
1. Magnetic Bead-based Enzyme-linked Immunosorbent Assay (MB-ELISA)
Enzyme linked immunosorbent assay (ELISA) refers to a method that binds soluble antigens or antibodies to solid-phase carriers (such as polystyrene) and performs qualitative and quantitative detection for them. According to the color change after the enzyme acting on the substrate, the result is analyzed and judged by the naked eyes or an enzyme-labeled detector. ELISA is one of the most popular methods due to its low-cost, high sensitivity and high throughput. However, microplate-based ELISA (including preceding coating, blocking, prolonged incubations and multiple washing steps) is always time-consuming. As an alternative, magnetic beads are always selected as the immobile phase to avoid preceding coating, blocking and reduce the incubation time. Therefore, magnetic bead-based ELISA (MB-ELISA) shows its superiority compared with the conventional ELISA, which not only has the advantage of high throughput, but also reduces the incubation time.
2. Magnetic Bead-based Chemiluminescence Immunoassay
Chemiluminescence (CI) is an optical radiation phenomenon produced by chemical substances in specific chemical reactions, and the emitted photons can be captured and analyzed by certain optical signal measuring instruments. Chemiluminescence immunoassay refers to the use of chemiluminescence reagents to label the antigens or antibodies, then go through a series of immunoreactions with the analytes, and finally determine the content of the analytes by measuring the luminescence intensity. This state-of-art technique is a combination of high-sensitivity chemiluminescence assay technology and high-specific immune response, which is widely used for the detection and analysis of antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs, as well as metal ions. Currently, magnetic beads are often chosen as the solid-phase materials in this system, possessing many advantages over conventional solid-phase microplates that are 96-well microtitration plates prepared with polystyrene. For example, magnetic beads can provide a large surface area, which allows for the utilization of a higher concentration of capture antibodies to enhance the sensitivity of the assay.
3. Magnetic Bead-based Fluorescence Immunoassay
Fluorescence immunoassay technology labels the antibody/antigen with fluorescein (fluorescein isothiocyanate or rhodamine, etc.) and uses a fluorescent microscope to observe the fluorescence generation or fluorescence intensity, therefore researchers can determine the presence, location and distribution of the antigen/antibody. This technique has the advantages of strong specificity, high sensitivity, and good practicability. It is often used to measure low-content biologically active compounds, such as proteins (enzymes, receptors, antibodies), hormones (steroids, thyroid hormones, phthalate hormones), drugs and microorganisms, etc. Magnetic beads (MBs), as a well-known hard magnetic material with very high magneto crystalline anisotropy, high coercivity and moderate saturation magnetization, have been successfully applied as matrices for the electrochemical detection of biomarkers. Therefore, the functionalized MBs could be used as fluorescence immunosensing probes in this method, realizing efficient removal of targeted antibodies from flowing biological fluids without washing steps by applying a magnetic field gradient.
4. Magnetic Bead-based Microfluidic Immunoassay
In clinical diagnoses, rapid analysis that offers point-of-care testing is required to assist the doctor to accurately diagnose and treat patients’ diseases or injuries. Microfluidics technology fits this requirement over other techniques like ELISA and flow cytometry. Microfluidic chips integrate the micro-channel network structure machine and other functional elements on a substrate within several square centimeters. The fluid in the micro-channel is controlled to achieve sample injection, dilution, mixing, reaction, separation and detection, having the advantages of miniaturization, integration, fast analysis speed, and low reagent consumption. Especially for magnetic bead-based microfluidic immunoassay, it has an edge over normal fluidic systems due to the unique merits of MBs mentioned above. However, currently there are still some problems that need to be solved. For instance, magnetic microbeads in fluidic devices are usually captured by external magnetic fields for collective measurement, which would create a mass of trapped beads that cannot be easily analyzed individually.
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