Diameter Microsphere size may be critical to the proper function of an assay, or it may be secondary to other characteristics. Considering traditional diagnostic methods, the test or assay format commonly dictates particle size, such as the use of very small spheres (~0.1-0.4µm) to ensure satisfactory wicking in lateral flow tests, or the use of larger, cell-sized spheres (~4-10µm) for bead-based flow cytometric assays. See our 300 series of application-specific TechNotes: 301, Immunological Applications; 302, Molecular Biology; 303, Lateral Flow Tests; and 304, Light-Scattering Assays.
In magnetic separations, particularly those involving capture and elution of target, the exact size of the magnetic particle may be unimportant, provided that the particles are in some general size range, and offer desired separation characteristics. See TechNote 102, Magnetic Microspheres.
Diameter also determines surface area. Small-diameter spheres present more surface area per unit weight, while larger spheres present more surface area per bead. Size also affects ease of handling, processing considerations (such as the method used for separations [centrifugation, dialysis, filtration]), and the amount of reagent needed for coating.
Common microsphere compositions include polystyrene (PS), poly(methyl methacrylate) (PMMA), and silica. These materials possess different physical and optical properties, which may present advantages or limitations for different applications.
Polymer beads are generally hydrophobic, and as such, have high protein binding abilities. However, they often require the use of some surfactant (e.g. 0.01-0.1% Tween® 20 or SDS) in the storage buffer to ensure ease of handling. During synthesis, functional monomers may be co-polymerized with styrene or methyl methacrylate to develop beads with surface reactive groups. Functional groups may be used in covalent binding reactions, and also aid in stabilizing the suspension.
Silica microspheres are inherently hydrophilic and negatively charged. Consequently, aqueous silica suspensions rarely require the use of surfactants or other stabilizers. Carboxyl- and amine-functionalized silica spheres are available for use in common covalent coating protocols, and plain silica microspheres may be modified using a variety of silanes to generate functional groups or alter surface properties.
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.
Many applications in the life sciences demand added properties, such as fluorescence or a visible color, or iron oxide inclusions for magnetic separations. Polymer spheres (and polymer-based magnetic spheres) are often internally dyed via organic solvent swelling, and many standard products are available. Dye concentrations can be adjusted to produce beads with different intensities to meet special needs, such as QuantumPlex™ for multiplexed flow cytometric assays, or our Dragon Green Intensity Standard, which supports imaging applications and associated instrument QC. Many surface- or internally-labeled fluorescent beads are also available as specialized flow cytometry standards.
Various types of superparamagnetic microparticles are also available – with different matrices, magnetite content, surface groups, etc. For new assays or applications, magnetic beads should be evaluated with the application demands in mind.