Immobilization

The first step in the interaction analysis is the immobilization of one of the interactants (ligand) on the sensor chip surface. This immobilization can be permanent in the form of a covalent bond or transient by means of capturing.

Below the topics, that needs attention during the immobilization procedure.

• Sample preparation: getting the ligand in the right purity and buffer.
• Choose an immobilization procedure depending on the ligand.
• How much ligand should be immobilized?
• Prepare the ligand (modifying techniques).
• Perform pre-concentration to determine the immobilization buffer and pH.
• The actual immobilization.

The immobilization or ligand coupling can be done in many different ways. Covalent coupling is stable and needs in general no modification of the ligand. The immobilization level is easily controlled and the ligand consumption is low. However, there is a reactive group needed (-NH2, -SH, -COH) on the ligand and the immobilization results often in a random orientation of the ligand.

The basic coupling procedures are the amine and thiol coupling. In addition, the (strept)avidin - biotin capturing is a very versatile procedure to capture proteins and oligonucleotides. The DNA mediated capturing of proteins is a specialized procedure for proteins, which are not easily coupled.

The coupling procedure consist in general of three basic steps:

• Activation: the dextran type surface has carboxyl groups which can be modified with NHS (N-Hydroxysuccinimide) and EDC (1-[3-(Dimethylamino)propyl]- 3-ethylcarbodiimide hydrochloride) resulting in an active group which can be further modified or used to couple the ligand.
• Coupling: by injecting the ligand over the activated surface until sufficient ligand is bound.
• Deactivation: by injection of a blocking solution like ethanolamine to quench remaining activated sites.