Before immobilization, the ligand has to be brought in a useful form and formulation. Most ligands can be immobilized without modification. However, sometimes a modification is required to use a specific immobilization technique. Some commonly used modifications are:
- adding a disulphide group
- adding a thiol group
- adding an aldehyde group
- adding a biotin group
The formulation of the ligand includes the buffer composition and possible additions. For most immobilization methods, a low salt buffer with a neutral pH is required (see pre-concentration). In addition, the buffer components should not interfere with the immobilization.
Adding a disulphide group
By modifying an amine group with NHS/EDC followed by PDEA a reactive disulphide group is introduced. This group can react with a thiol group, which is on the sensor chip surface (surface thiol method).
Adding a thiol group
By modifying an amine group with NHS/EDC followed by DTE and cysteamine a thiol group is introduced. This group can react with a reactive disulphide group, which is on the sensor chip surface (ligand thiol method).
Adding an aldehyde group
For polysaccharides and glycoconjugates aldehyde coupling is a good alternative for amine or avidin-biotin coupling. This is especially the case for molecules containing sialic acid, because these residues are easily oxidized to aldehydes.
Aldehydes can be introduced by oxidizing cis-diols with sodium metaperiodate resulting in an aldehyde group (1). This group can react with a hydrazide group introduced on the sensor chip surface and the formed hydrazone bond is stabilized by reduction with cyanoborohydride.
Adding a biotin group
For the biotinylation of macromolecules, several commercial kits are available that have their own instructions for use. A more thorough discussion of biotinylation can be found in (2). More about biotinylation of nucleic acids can be found in Methods in Molecular Biology vol. 4. (1988).
There are two important points to consider in context with biomolecular interaction analysis. The degree of biotinylation should be kept low (0.5 - 1.5 mole of biotin per mole of ligand) (3). At higher levels the binding kinetics are faster but the amount of ligand bound is reduced. Higher levels of biotinylation may help stabilize the immobilized ligand against regeneration conditions (4). Excess of free biotin must be removed efficiently. Gel filtration on a desalting column, dialysis or centrifugal ultra-filtration is recommended for medium-sized to large ligands (Mr > 5000 Da). New sensor chips with capturing methods for (strept)avidin-biotin complexes make this type of modification very flexible (3).
|(1)||O'Shannessy, D. J. and Wilchek, M. Immobilization of glycoconjugates by their oligosaccharides: use of hydrazido-derivatized matrices. Analytical Biochemistry 191: 1-8; (1990).|
|(2)||Wilchek, M. and Bayer, E. A. Introduction to avidin-biotin technology. Methods Enzymol. 184:5-13.: 5-13; (1990).|
|(3)||Papalia, Giuseppe and Myszka, David Exploring minimal biotinylation conditions for biosensor analysis using capture chips. Analytical Biochemistry 403: 30-35; (2010). Goto reference|
|(4)||BIACORE AB BIACORE Application Handbook. (1998).|