The sensor chip
The sensor chip in Biacore systems consists of a glass slide coated with a thin layer of gold. These components, together with the docking system for mounting the sensor chip in the optical system, are required for generation of a surface plasmon resonance (SPR) signal. To provide a suitable environment for the molecular interactions being studied, the gold surface is covered by a linker layer and (on most sensor chip types) a matrix of modified dextran. It is the surface matrix that determines the properties of the sensor chip with respect to ligand attachment and molecular interaction.
How is the ligand attached to the sensor chip surface?
There are two main approaches to attaching macromolecular ligands to the sensor chip surface, covalent immobilization and high affinity capture.
Covalent immobilization involves irreversible chemical attachment of the ligand to the surface, usually to the carboxymethyl groups on CM-series sensor chips. The ligand remains on the surface throughout the lifetime of the sensor chip, and is regenerated to remove non-covalently bound molecules after each analysis cycle.
Covalent immobilization may exploit amine, thiol (either native or introduced) or aldehyde groups (obtained by oxidation of cis-diols) on the ligand. Amine coupling is the most widely used alternative.
High affinity capture
High affinity capture refers to attachment of ligand by binding to an immobilized capturing molecule, and offers advantages over covalent immobilization in a number of situations:
- Capture does not require any chemical modification of the ligand and can be performed in physiological buffer conditions. It can therefore be used with ligands that are not amenable to covalent immobilization.
- Normally, regeneration of the surface with a captured ligand involves removal of ligand together with other bound molecules, leaving the capturing molecule ready bind fresh ligand for the next cycle. While this procedure increases consumption of ligand, it removes the requirement that ligand is not damaged by regeneration and allows the ligand to be changed between analysis cycles on the same sensor chip.
- Capture may be used to attach a specific ligand from a complex sample, provided the capturing interaction is sufficiently selective. Covalent immobilization of ligand from such samples would require prior purification of the ligand.