The first step in conducting a study with Surface Plasmon Resonance is to immobilize the ligand of interest on the surface of a gold sensor chip, regardless of whether you are investigating fusion proteins, protein-DNA binding, antibodies, peptide chains or some other macromolecule. There are two broad approaches for performing this immobilization: chemical coupling (i.e., forming a covalent bond) and capture (i.e., forming a non-covalent bond). Furthermore, there are multiple chemistries that can be used to accomplish each type of approach. Deciding which strategy you should use is dependent on the ligand you are immobilizing and the analytes you plan to assay. This Surface Plasmon Resonance Insider post will help to shed some light on the different options that are available as you plan your SPR experiment.

Chemical coupling strategies result in a covalent bond between the ligand and the sensor chip’s surface. The most common chemistry employing this strategy is amine coupling, which is utilized in the majority of published SPR studies. Other coupling chemistries include thiol, aldehyde and maleimide. The ease, simplicity and variety of chemistries available for performing direct coupling are only a few of this approach’s advantages. Covalently binding the ligand also forms a stable surface on the chip, and if multiple points of binding are available then a surface with very high ligand density can often be prepared.

However, there are limitations to the direct chemical coupling approach. The main disadvantage is that some of the ligand molecules may attach to the chip in random orientations, which renders many of the ligand’s binding sites inaccessible to the analyte. Another drawback is that amine coupling requires a low pH environment, which can potentially denature or deactivate the ligand and yield low analyte responses.

Alternatively, you can “capture” a ligand over a suitable surface. Common capturing surfaces are a nickel-NTA chip for capture of His-tagged proteins, a streptavidin or NeutrAvidin chip for capture of biotinylated molecules, a hydrophobic chip for capture of lipids, anti-Fc antibody capture, and protein A capture of IgG antibodies. This strategy typically involves the capture of ligands in a specific orientation, which ensures that the ligand’s binding site is more readily available for binding. Since capture methods do not require a lower pH for capture of the ligand, ligands remain active because of this milder environment.

Capture strategies do have some disadvantages as well. In general, the bonds created by the “capture” are not as strong as covalent bonds. These weaker bonds can create an unstable surface, and ligands may dissociate from the surface. Also, the capture approach typically consumes more ligand as the ligand needs to be recaptured after each regeneration step. An exception to this drawback is the biotin-streptavidin method, which captures ligand with bonds that are nearly as strong as covalent bonds. Lastly, the capture method results in a lower ligand density on the sensor surface as there is typically only one available site on the ligand to interact with the sensor surface.

We hope you found this post helpful and informative. You can read more about SPR elsewhere on Reichert’s website, including our first blog post and information on the technology of SPR.

If you would like advice on determining the most appropriate immobilization strategy for your application, please do not hesitate to contact us. Also, please contact us if you would like to suggest topics for future blog posts or if you have any suggestions to make this column even better.