Microsphere Handling
Coating
Microspheres may be coated with capture molecules, such as antibodies, oligonucleotides, peptides, etc. for use in diagnostic or separation applications. Microsphere coatings are typically optimized to achieve desired specific activity while minimizing nonspecific interactions. Consideration should also be given to required stability, development timeframe and budget, and the specific biomolecule to be coated. These factors will aid in determining the most fitting coating strategy for both short- and long-term objectives. Standard microsphere products support three basic coating strategies: adsorption, covalent coupling, and affinity binding.
Adsorption relies primarily on hydrophobic interactions between the biomolecule and the polymer particle. Such coatings are fairly simple to conduct, involving the incubation of microspheres with the purified biomolecule. They typically require little optimization, and reagents may be developed relatively quickly. However, as adsorption relies on the formation of multiple attachment points between the molecule and the particle, this strategy is typically reserved for use with proteins and non-functionalized polymer spheres. Adsorption is generally not suitable for hormones, peptides, or nucleic acids in hybridization-based applications, and protein adsorption to silica is expected to be less efficient than to polymer. See TechNotes 201, Working with Microspheres, and 204, Adsorption to Microspheres.
Covalent coupling results in the permanent attachment of the molecule to the functionalized (e.g. carboxyl or amine) microspheres. It can provide needed stability when developing a commercial reagent, and for multiplexed assays, where analyte-specific bead populations are mixed. Additionally, specialized chemical linkers may be employed to address steric effects or to optimally orient the molecule. Although covalent binding protocols often involve a higher level of optimization than other approaches, coupling kits are available to simplify the process. See TechNotes 201, Working with Microspheres, and 205, Covalent Coupling.
Affinity binding is a straightforward method for immobilizing primary antibodies or biotinylated molecules. Proteins A and G and Fc-specific antibody coatings permit the directed immobilization of primary antibodies, and streptavidin is used extensively for the binding of biotinylated molecules, such as antibodies, peptides, and oligonucleotides. See TechNotes 101, ProActive® Microspheres, and 302, Molecular Biology.
It is important to note that each binding strategy has benefits and limitations, which should be weighed in the context of study objectives and the demands that will be placed on the finished reagent.
Washing
Microspheres sold as instrument standards can often be used as-is, or simply diluted in an appropriate buffer or aqueous solution. Conversely, microspheres that will be coated or otherwise modified should be washed to remove additives and residuals that could interfere with binding reactions or other processes.
Common washing and separation methods for non-magnetic beads include centrifugation, filtration, and dialysis. Selection of the "best" method will depend upon scale, required throughput, and microsphere characteristics. Centrifugation is often used for small spheres <0.5µm, or to achieve higher throughput. Superparamagnetic microparticles are separated using rare earth or electro-magnets. See TechNote 203, Washing Microspheres.
Aggregation
Our microspheres are available in a variety of compositions, including polystyrene, poly(methyl methacrylate), and silica. Though polymer microspheres are more susceptible to hydrophobic-mediated aggregation, there are several factors that may influence dispersity of the suspension. For example, low surface charge, small diameter (high surface area:volume ratio), high microsphere concentration, and suboptimal buffer composition or pH may promote aggregation. Strategies that are effective in addressing aggregation thus counter these conditions, i.e. use of surfactant to reduce hydrophobicity (e.g. 0.01-0.1% Tween® 20 or SDS), sonication to disrupt aggregates, and adjusting microsphere concentration or buffer pH to deter contact between individual spheres. See TechNote 202, Microsphere Aggregation.