The first step in planning your Surface Plasmon Resonance (SPR) experiment is choosing the best sensor chip for the job. There are a variety of chips to choose from, but they can be classified into two main groups: 2D (planar) and 3D (hydrogel). Each type of chip has its own advantages depending on what type of experiment you plan to run. This Surface Plasmon Resonance Insider post will provide you with the information you need to help you decide which type of chip to choose as you plan your SPR experiment.
Simply put, the surfaces of planar chips consist of parallel single-chain molecules, each of which is designed to bind with a single analyte molecule (see Figure 1). The surface backbone is based on self-assembled monolayer chemistry with 10% of the terminal residues bearing COOH groups and the remaining groups being polyethylene glycol, which minimizes non-specific binding. Planar chips are ideal for experiments where the binding between two macromolecules is measured. Examples are protein–protein interactions, antibody–protein interactions, protein–carbohydrate interactions, and so forth. A planar chip surface has a much lower binding capacity compared to a 3D surface, which limits its functionality for low molecular weight detection. However, distinguishing features of planar surfaces are that ligand crowding is much less of a problem, and analyte re-binding is less likely during the dissociation phase.
Figure 1: Reichert’s monothiol mixed self-assembled monolayer (SAM) 2D planar chip, which binds one analyte per ligand.
To bind a smaller analyte to a large, immobilized molecule (e.g., for small molecule drug discovery against a macromolecule target) you will want to use a 3D chip. The surfaces of 3D chips are about 100-fold thicker than planar chips and consist of a hydrogel polymer. Dextran that has been carboxymethylated is the most common 3D chip used for SPR. A ligand can bind to multiple sites on the hydrogel matrix, which can significantly increase the amount of ligand that can be coupled to the surface (see Figure 2). Dextran hydrogel polymers have additional advantages that make them a popular choice for SPR analyses since they exhibit low non-specific binding, present a very stable surface, and provide a “solution-like” environment for the ligand. Dextran chips are not solely limited to small molecule analysis, as they are also commonly used for protein–protein interactions, but caution must be exercised to not crowd the surface with a high ligand density.
Figure 2: Reichert’s carboxymethyl dextran polymer 3D chip can bind to significantly more analytes than a 2D planar chip.
Whether using a planar or hydrogel chip, immobilizing the right amount of ligand is critical because the analyte binding responses are proportional to both the analyte molecule weight and the amount of ligand immobilized, and inversely proportional to the ligand molecular weight as shown in the following equation:
For kinetics experiments, you typically want to estimate how much ligand to immobilize before starting your experiment. Base your starting experiment using an initial estimated analyte binding response from the above equation of about 50 to 100.
Both 2D and 3D chips can be used for direct coupling via an amine, thiol or malemide group. Capture experiments via Ni-NTA (for His-tagged proteins) or NeutraAvidin or Streptavidin (for biotinylated molecules) can also be carried out using either a 2D or 3D chip. But, keep in mind that if you want to prepare your own NeutrAvidin or Streptavidin chips using a 3D surface then you will need to carefully control the amount of material coupled to minimize crowding on the 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 an earlier blog post on immobilization strategies (direct coupling vs. capture methods).
If you would like advice on determining the most appropriate chip 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.