IDC: Working with Microspheres


Working With Microspheres *Introduction* IDC latex microspheres are available with either anionic (negative) or cationic (positive) surface charges. Anionic latexes – those with sulfate, carboxyl, or carboxylate modified surface groups – are less likely to bind to negatively-charged cell surfaces and are therefore used most frequently in biological applications. Every possible precaution is made throughout the manufacturing process to ensure that the particles are kept free of contaminants. All handling and packaging of our latex products are carried out in our clean room facility. The final product is sold as a suspension in ultra-pure water. If you wish, you may sterilize the latex prior to proceeding with your application by one of the following methods: Pasteurization: 24 hours at 78-80º C Gamma irradiation: 0.03 megarads for 24 hours *Surface Properties* Even though the particles have charged surfaces, the hydrophobic latexes will bind strongly to any molecule that has a hydrophobic character, including proteins, nucleic acids, and many small biomolecules such as drugs and hormones. Hydrophobic latex products are usually stable in systems free of biological molecules without further modifications. In biological systems however, including applications for immunoassays, the microspheres can easily be coated with various proteins or polysaccharides that will greatly reduce their capacity to absorb biomolecules non-specifically. Specific irreversible adsorption of protein molecules such as avidin, streptavidin, and antibodies is accomplished by simply mixing the latex and protein together for a specified period of time, then separating the bound from the unbound protein through centrifugation and removal of the supernatant. To reduce nonspecific binding, the particles can be coated with BSA or dextrans. To further reduce nonspecific binding, proteins, nucleic acids, and other biomolecules can be covalently coupled to the particles. Covalent coupling requires more effort than passive adsorption, but can result in conjugates with greater specificity that remain active longer. Carbodiimide-mediated coupling to CML latexes is the method of choice for conjugating low molecular weight peptides and oligonucleotides. If you're interested in learning more about using microspheres in your applications, contact us and ask for information about our Latex Lectures. *Choosing the Right Buffers* Choosing the right buffer is critical when working with latex microspheres. Consider the following points when choosing a buffer: The choice of buffer is dependant on the type, size, and density of surface charge groups. In general, smaller particles have fewer surface charge groups for stabilization, and therefore need more stringent conditions to prevent aggregation. When using sulfate, carboxyl, or CML (negatively-charged) latex particles, cationic buffers such as Tris should be avoided. Negatively-charged particles are also sensitive to low concentrations of multivalent cations such as Ca⁺⁺ and Mg⁺⁺ salts, therefore buffers containing these cations should be avoided if at all possible. If multivalent cationic buffers are necessary for an experiment, then cationic latex particles which are not as sensitive to Ca⁺⁺ and Mg⁺⁺ ions are a better choice. When using cationic (amidine and AML) latex particles, phosphate or borate buffers should be avoided. The ionic strength of the buffer should be kept as low as possible, especially when the particles are very small or when they have a low charge density. Buffer pH is also important when using carboxyl and CML microspheres. The particles should be used at a pH > 5.0. If these conditions are not followed, the charge groups may be neutralized, leading to aggregation. If aggregation does occur, the particles can usually be redispersed by adjusting the pH to the correct range, followed by gentle sonication. Finally, the water used in buffer preparation should be as pure as possible to prevent scavenging of impurities by the hydrophobic particles. *Controlling Non-Specific Binding of Proteins to Latex Particles* Nonspecific binding is probably the most common problem encountered in working with latex particles, and is often the major reason for abandoning an otherwise well-conceived experiment. Latex particles are generally hydrophobic, and although various modifications tend to make them less so, the particles always retain some hydrophobic characteristics because they are polystyrene-based. In biological systems, most of the nonspecific binding problems are a result of hydrophobic interactions. However some of the problems may also be caused by charge-based interactions, such as a positively charged molecule binding to a negatively-charged latex surface. The best way to minimize these nonspecific binding events is to coat the particle with a large molecule such as a protein or polysaccharide which reduces nonspecific binding by blocking the hydrophobic or charged binding sites on the particle surface. Although many types of blocking agents may be used, the most frequently used are BSA, egg albumin, and whole serum. Egg albumin should be avoided in biotin-avidin systems. When using hydrophobic latexes, add the desired protein at a concentration of 250-500 µg/mL of latex suspension at a concentration between 0.5 and 2.0 per cent solids. Dextran (40k MW at 2% w/v) can be used as a blocking agent in place of, or in addition to proteins. Unlike proteins, dextrans bind reversibly to latex microspheres, to form a layer on the surface, creating a more hydrophilic environment and reducing nonspecific interactions. When a hydrophilic CML particle is used, the blocking agent may not bind as strongly. In this case, covalently coupling the protein may solve the problem. Specific binding proteins, such as immunoglobulins or avidin can be mixed with BSA and simultaneously coupled covalently, resulting in a specifically active latex with covalently-bount BSA coating. In situations where detergents are acceptable, a nonionic surfactant such as Triton X-100 or Tween 80 can be coated onto the particle at concentrations ranging from 0.01 to 0.1%. The exact concentration should be determined experimentally for each application.