This is the second of a series of posts on pros and cons of classical covalent links, which we will publish during the next few weeks. This one is about 3 well-known classical covalent links: tosyl, epoxy and chloromethyl groups. In the next posts we are going to review other examples, such biotin or streptavidin beads.
Magnetic latex particles can be covalently attached to a protein through functional groups present on the surface of the particle. There are a number of linkages to choose from, as beads with different types of coatings are commercially available. Some of these surface functional groups require activation before they can bind a molecule. Others, such as tosyl, epoxy, and chloromethyl groups, do not require any preliminary activation. The advantage of these types of surface groups is that they will attach a molecule as long as the conditions (pH, temperature) are favorable for binding.
This post is about classical covalent links for biomarkers. If you want detailed information about this topic, download our free ebook The Advanced Guide for the use of Magnetic Beads in Chemiluminescent Immunoassays:
What are the necessary conditions for these groups to bind?
Tosyl groups, for instance, will bind to amino and sulfhydryl groups in a protein at a neutral pH and a temperature of 37ºC. Epoxy groups can bind a number of different groups, depending on the pH of the binding reaction. If the pH is slightly basic, epoxy groups will bind thiol groups. At higher pH conditions, the epoxy group will bind to amino groups. Finally, at very high alkaline conditions, epoxy groups can bind to hydroxyl-containing ligands. Of the three groups listed previously, however, the easiest to work with is the chloromethyl group. These groups will bind amino groups at room temperature and at a neutral pH.
The advantage to using beads containing the covalently bonding tosyl, epoxy, and chloromethyl surface functional groups is that functionalizing the beads is more straightforward. There is no preliminary activation that needs to be carried out prior to attaching a molecule to the bead. The binding, however, occurs with a lesser degree of specificity than is seen with covalent bonding functional groups that require activation, such as carboxyl, amino, and hydroxyl groups, and some passive adsorption may occur.
Don't forget to check these posts from our blog in order to get a deeper insight into magnetic particles and immunoassays:
- Using streptavidin magnetic beads in Chemiluminescent Immunoassays
- Is There any Advantage of Using Beads with Plain Coatings?
- Pros and Cons of Classical Covalent Links (-COOH, -OH, -NH2)