Magnetic bead technology has developed rapidly in the past decade. New functionalization strategies are continually hitting the market as more laboratories begin to use magnetic separation systems to identify and isolate cells and microorganisms. There is a direct correlation between advanced functionalization strategies and improved assay selectivity. The size of magnetic beads (μm-nm) places them squarely into the cellular realm, and their surface functionalization causes them to bind to specific surface ligands. This functionalization step is crucial to the separation process because it allows the operator to control which cells the beads will bind to. For a positive-selection immunoassay, the bead surfaces are functionalized by covalent attachment of an antibody that will bind to the cell or micro-organism of interest. This bond ensures that the microbe-bead conjugate will be captured in a magnetic field gradient.
New developments have introduced modified bead surfaces (carboxyl, amino, hydroxyl, and sulfates), pre-activated surfaces (tosyl, epoxy, and chloromethyl groups), and bio-activated surfaces (protein A, protein G, streptavadin-biotin). Firstly, modified bead surfaces provide a way to covalently attach surface antibodies, and can be quite useful. However, functionalization of these surfaces must be done in a non-polar solvent and aggregation and non-specific binding can be an issue. Many of the sites are common to multiple proteins and locations within a single protein.
Pre-activated bead surfaces such as tosyl, epoxy, and chloromethyl groups add a level of control through manipulation of solution pH. Tosyl actively binds to sulfhydryl groups at a neutral pH, but switches to amino groups as the solution becomes more basic.
The most expensive, but most specific option is to use bio-activated surfaces of protein A/G and streptavadin-biotin affinity ligands. Streptavadin has an incredible specificity for biotin, and the affinity between the two is unaffected by changes in pH, salt concentration, or the presence of detergents. This specificity can be very useful. Another layer of control emerges in the fact that the link is reversible if the streptavadin-biotin conjugate undergoes a short 70°C incubation. This feature introduces an easy method for the separation and removal of magnetic beads following target isolation.
All of these surface functionalization methods provide control over specificity and stability of the bead-target coupling, but they don't address functional group orientation or density. The functionalization strategies above are enacted by incubating the functional moieties in molar excess with the magnetic beads. This can result in the attachment of functional groups upside-down or at angles that decrease target binding efficacy. Similarly, with such an excess there will be a high density of functional groups in multiple layers on one bead, and steric effects can block target-binding access. Current and future solutions to this problem have arrived in the form of:
- Improved protocols for magnetic bead functionalization
- A deeper understanding of the kinetics of antigen capture
- Development of new conjugation chemistry.
This new conjugation chemistry consists of new polymeric metals ions and coordination complexes to improve control over ligand density and orientation on a variety of surfaces. More detailed information and references to this research can be found within the SEPMAG ebook entitled Magnetic Bead Coatings Today and Tomorrow.
- A new microfluidic technique to create fluorescent and magnetic Janus particles
- Magnetic Nanoparticles
- Immunomagnetic cell separation