2.2. Immobilization Strategies

The immobilization of a protein ligand over a sensor surface is based on two main strategies; one involving a direct binding of the ligand to the surface by covalent coupling, the other using an indirect immobilization through the high affinity capture of the ligand by a covalently coupled molecule. There are three main types of covalent coupling chemistry, using the amine group of lysines, the thiol group of cysteines or the aldehyde group of carbohydrates, to covalently bind proteins to sensor chip surfaces exposing free carboxymethyl groups, such as CM-series chips (GE Healthcare). The most used chemistry is the amine coupling which consists of the activation of the carboxymethyl groups by N-ethyl-N-(3-diethylaminopropyl) carbodiimide (EDC) and N-hydroxyl-succinimide (NHS) to give reactive succinimide esters, which spontaneously react with protein amines to form covalent links. An alternative strategy, using bare gold SPR sensor disks, involves the gold surface functionalization with cysteamine and glutaraldehyde, prior to the covalent binding of proteins through amine coupling. These direct immobilization approaches don’t need any ligand modification but cause the immobilization of the ligand in different orientations. Some of these orientations may have a negative effect by decreasing or even abrogating the ligand ability to bind to the analyte. In addition, an efficient ligand regeneration could be difficult to be achieved. On the other hand, in the indirect immobilization strategy, the ligand needs to have a suitable binding site or a tag allowing it to be captured with a high specificity and to be effectively dissociated by regeneration procedures. The most used strategies involve antibody capture of tags, such as GST (e.g., GST Capture kit, GE Healthcare), usually linked to the N-terminus of recombinant proteins. This strategy has important advantages since proteins are rarely inactivated by indirect coupling and all the molecules are immobilized in a known and well determined orientation on the surface. In addition, by using appropriate buffers, the captured ligand-analyte bond can be selectively dissociated, thereby enabling the surface to be re-used. In any case, a control surface should be generated, being as similar as possible to the ligand surface, to measure non-specific binding and to record the background response.