Common Types of Magnetic Beads and Considerations for the Coating Process

According to the different functional groups, the types of magnetic beads are diverse. Commonly used ones include carboxyl magnetic beads, N-acryloyl amine magnetic beads, and streptavidin magnetic beads. Carboxyl magnetic beads are widely used in enzyme-linked immunosorbent assays (ELISA), while streptavidin magnetic beads are more commonly used in Roche electrochemiluminescence assays. Streptavidin magnetic beads are usually composed of three components: biotin-labeled antibodies, magnetic beads, and luminescent-labeled antibodies, which are combined in a kit. Compared to other types of reactions, the reaction with streptavidin magnetic beads is milder. The diameter of the magnetic beads is a crucial parameter as smaller beads can bind more antibodies, thereby increasing the surface area for antibody binding and enhancing the efficiency of antigen-antibody binding.

1. Carboxyl Magnetic Beads:

Carboxylation is the most common surface modification method for nanomaterials. By modifying the surface of magnetic beads with carboxyl groups, the hydrophilicity of the beads is increased, improving their stability in aqueous solutions and effectively reducing nonspecific adsorption. Carboxylated magnetic beads have abundant carboxyl groups on their surfaces, allowing proteins, nucleic acids, peptides, and other molecules to be coupled to the beads through specific chemical reagents. They are widely used in protein purification, immunoassays, molecular detection, NGS, and cell separation.

In chemiluminescent immunoassays, magnetic beads serve as reaction carriers, with specific antigens or antibodies immobilized on their surfaces to capture target molecules in the sample. Proteins can bind to carboxyl-modified particles through adsorption, which is mediated by hydrophobic and ionic interactions between the protein and the bead surface, and the adsorption occurs very rapidly. In addition to adsorption, proteins can also be covalently linked to the carboxyl-modified bead surface. Carboxyl groups can be activated by water-soluble carbodiimides (e.g., EDC) to react with the free amino groups on the adsorbed proteins, forming amide bonds. Hydrophobic interactions and covalent bonding are the main mechanisms involved. Tris buffer systems are commonly used during the coating process.

The commonly used method for coating carboxyl magnetic beads with antibody proteins is the EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) activation method. EDC is a water-soluble small molecule that does not require organic solvents for dissolution. The byproduct of the reaction is also water-soluble and can be easily removed by water washing or dialysis. The activated carboxyl groups can form stable amide bonds with the amino groups of antibodies, thereby immobilizing the antibodies on the bead surface. This reaction is usually performed in a slightly acidic environment.

There are two methods of EDC-activated carboxyl coupling protein. One method is the one-step method, where the antibody and EDC are added to the carboxyl magnetic bead solution sequentially. In this reaction, EDC activates the carboxyl groups and reacts with the amino groups of the antibody simultaneously. This method is simple and convenient, but an excess of EDC can lead to antibody aggregation and reduced activity. Moreover, the activated ester intermediate formed by EDC activation of carboxyl groups reacts slowly and is prone to hydrolysis, resulting in low reaction efficiency.

The other method is the two-step method, which separates the EDC activation of carboxyl groups and the reaction with protein amino groups into two steps. This method usually involves introducing Sulfo-NHS activated esters during the first step of carboxyl group activation. Sulfo-NHS (N-hydroxysulfosuccinimide) is a hydrophilic small molecule that can quickly react with amino groups. After EDC activates the carboxyl groups, a stable intermediate is formed with Sulfo-NHS. After removing the excess EDC, the antibody protein is added, and the amino groups on the protein react with the NHS intermediate to form amide bonds. The two-step method effectively avoids antibody aggregation caused by residual EDC and improves the efficiency of amide bond formation. Some studies have shown that the two-step method with Sulfo-NHS can achieve 20 times higher protein coupling efficiency than using EDC alone. In addition, the sulfonic acid groups of Sulfo-NHS can form a negatively charged layer on the bead surface after carboxyl activation, maintaining the stability of the bead surface and preventing bead aggregation.

2. Tosyl Magnetic Beads:

Tosyl groups are the most widely used surface modification for chemiluminescent immunoassays. Tosyl groups can covalently bind to antibodies and other molecules without the need for pre-activation with coupling agents. The reaction is highly controllable, with low non-specific adsorption and good reproducibility during the coating process, making it suitable for large-scale production of magnetic beads.

Brands such as Dynabeads, JSR, and Merck produce tosyl magnetic beads. Due to the more complex surface modification process, there are few domestic magnetic bead manufacturers that prioritize the development of tosyl-modified beads.

Since tosyl-coated beads do not require a coupling agent, molecules with amino groups, such as antibody proteins, can be chemically immobilized on the bead surface. As the chemical binding progresses, the tosyl groups gradually dissociate, increasing the hydrophilicity of the particle surface, maintaining the bioactivity of the bead conjugate, and effectively suppressing non-specific reactions during analysis.

In buffer systems with pH ranging from 7.0 to 8.0, the tosyl groups react with thiol groups on proteins. When the reaction buffer pH is adjusted to 8.5 to 9.5, the reaction occurs with the amino groups on the protein.

During the coupling of antibodies, a 100 mM borate buffer is commonly used as the reaction solution. The magnetic beads are washed with the borate buffer two to three times and then resuspended in the buffer using ultrasonication. The concentration of tosyl groups in the coating process is generally higher than that of carboxyl groups. A concentration of 30 mg/mL is typically used for coupling and coating. After mixing the magnetic beads and proteins, the reaction tube is placed in a thermostatic shaker at 37°C overnight (12-18 hours) for the antibodies to react with the tosyl groups. If a stable reaction is not achieved, the reaction time needs to be extended. After the coating process, the beads can be sealed using a PB buffer solution containing 0.5% BSA at pH 7.4. The sealing process usually takes 1 hour at 37°C or can be performed overnight at 4°C. The beads can be stored in a working buffer solution, typically at a concentration of 10 mg/mL.

3. Streptavidin Magnetic Beads:

Streptavidin is a tetrameric glycoprotein that can bind to the small molecule biotin with one subunit per molecule. The binding between streptavidin and biotin is one of the strongest known non-covalent interactions. Generally, streptavidin is immobilized on the bead surface through covalent coupling. Antibodies are then biotinylated, and the antibodies are connected to the bead surface through the specific binding between biotin and streptavidin. This method allows for the detection of multiple analytes using a single type of magnetic bead. Roche Diagnostics uses the biotin-streptavidin system for nearly all chemiluminescent assays.

Streptavidin-coated magnetic beads can easily bind to biotinylated biomolecules, allowing for probe preparation or separation of biotinylated biomolecules. Coupling CRP monoclonal antibody to streptavidin magnetic beads and using them in immuno-chromatographic test strips is an example. Streptavidin magnetic beads linked with biotin-antibody conjugates can specifically absorb biotinylated molecules with high purity from samples. The hydrophilic polymer coating on the bead surface does not interfere with enzyme-catalyzed reactions, and the addition of beads to PCR reaction systems does not affect nucleic acid amplification. In enzyme immunoassays, they can also be used as carriers for biotinylated antibody markers.

4. Coating Experience:

The commonly used ratio for coating magnetic beads with antibodies is 20 μg/mg. The concentration of the magnetic bead stock solution is 10 mg/mL. The working solution is typically a neutral buffer solution diluted at 1:50, resulting in a final concentration of 0.2 mg/mL. Aggregation of magnetic beads may occur during the coating process. In this case, the processes for handling the beads and antibodies or the quality of the antibody raw materials should be considered. Most projects do not experience aggregation, but projects involving coating magnetic beads with antigens may encounter aggregation. In such cases, the buffer system should be screened or the raw materials should be purified through methods such as dialysis.