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Activating the surface

Regardless of the chemistry used to attach the ligand to the sensor surface, the first step in covalent immobilization procedures is activation of the carboxyl groups on the surface with a mixture of 0.2 M EDC and 0.05 M NHS to give reactive succinimide esters.

Activation of carboxymethyl dextran with EDC/NHS. The dextran chains are omitted from this and similar illustrations for simplicity.

The succinimide esters react spontaneously with amine and other nucleophilic groups, allowing direct immobilization of molecules containing such groups. Amine coupling is the commonest form of this kind of immobilization reaction for proteins and for a range of other molecules.

Other groups can be introduced on to the dextran matrix once it has been activated with EDC/NHS. One commonly used example is the introduction of reactive disulfides that can then be used in a thiol-disulfide exchange reaction to immobilize thiol-containing ligands (ligand thiol coupling). Another is the introduction of hydrazide groups which can react with cis-diols obtained by oxidation of aldehyde-containing molecules.

The well-defined chemistry of the dextran matrix and of EDC/NHS activation allows alternative approaches to covalent immobilization to be devised for situations where the more standard methods are not suitable.

See also

List of help files

Covalent immobilization