Getting ready to plan for your surface plasmon resonance (SPR) experiments? Great! So, you probably already know the value of using SPR technology and how it is becoming more affordable and accessible for researchers everywhere. We believe that SPR will become a standard technique available in every biochemistry lab. If you have yet to discover the advantages of surface plasmon resonance, visit our blog post 6 advantages of surface plasmon resonance. The first step in planning for your SPR experiment is to determine which type of SPR sensor chip is right for your application.
The type of sensor chip required depends on your ligand, which needs to be immobilized to the sensor chip before you can measure the interaction between your ligand and analyte. There are two categories – covalent or capture coupling-based sensor chips. Today, you will learn the differences between the two and the advantages and disadvantages associated with both.
The Types of SPR Sensors and their ‘Personalities’
Sensor chips consist of a glass substrate, nanogold coating, and usually a functional chemical coating on the gold surface. For sensors to work, they require the immobilization of ligands to the gold surface, and each type of sensor chip comes with its own personality (advantages and disadvantages) depending on whether you choose covalent or capture based coupling, and the surface chemistry used. Fortunately, there are generally inherited benefits to using SPR sensor chips such as reusability which lowers the cost of consumables. The number of times you can reuse the sensor chip with the ligand attached depends on the stability of the ligand. Here is our list of coupling sensor chips at Nicoya Lifesciences:
Covalent coupling sensor chips:
- Gold – no surface chemistry
- COOH – for coupling to any amine group on the ligand
- Amine – for coupling to activated carboxyl groups on the ligand
Capture coupling sensor chips:
- Streptavidin – for coupling to biotinylated ligands
- Biotin – for coupling to streptavidin tagged ligands
- NTA – for coupling to his-tagged ligands
Sensor chip containing ligand (binding partner attached to the surface) and free analyte (binding partner in solution)
Covalent Coupling – SPR Sensor Chips
With plain gold sensor chips, you can immobilize the ligand in either of two ways: direct immobilization and thiolation. Direct immobilization is possible using the weak interactions that may exist between the ligand and the sensor surface, but this is not recommended for most applications. Why, you may ask? – because it is not stable or repeatable and could degrade the ligand. A third option for covalent coupling is using EDC/NHS chemistry to directly bind to the ligand through its amine and carboxyl group.
1. Gold Sensor Chips – The Malleable Personality
The simplest immobilization method is direct immobilization. The benefit to this immobilization technique is its simplicity as it does not require any extra chemicals to facilitate the immobilization. The drawback, however, is the inability to control the degree of functionalization of the ligand, not to mention the orientation and stability of the ligand as well. The consequential result is the uncertainty of replicating experimental results. To further complicate the situation, protein-based ligands with lysine or cysteine groups will have a tendency to bind to the surface more readily than other amino acid residues. This is due to the N-termini of proteins, amine groups and thiol groups, that can actively bind to a gold surface which ultimately prevent it from binding to analytes.
Thiolation on the other hand, is the more commonly used method to immobilize ligands directly to a gold surface. The thiol group, either native to the ligand or introduced specifically for this purpose, will form a strong bond with gold surfaces spontaneously. To block any uncoated surfaces, short thiolated PEG molecules and BSA can be used after the immobilization of the ligand. The immediate benefit of this method is that it allows the ligand and analyte to bind close to the gold surface. This enhances binding signal due to greater localized refractive index shift.
Takeaway: users can develop their own surface chemistry through the use of the plain gold sensor chips. This provides an ideal platform for customized surface chemistry or for providing a rich teaching environment in undergraduate labs.
2 & 3. COOH and Amine – Fraternal Twins
Well, what about the COOH and Amine sensor chips? Both sensor chips require EDC/NHS chemistry to activate the carboxyl groups and amine groups which can then bind to the ligand through its amine and carboxyl group, respectively. Fortunately for you, we put together a well-crafted activation kit which contains all reagents necessary to perform this coupling and has been optimized for best performance.
Covalent coupling of ligand to COOH sensor chips using EDC/NHS activation
To get your very own kit containing all reagents necessary to perform this coupling, check out Nicoya’s Activation Kit.
One of the enjoyable features of this coupling method is that it’s relatively straightforward to perform with good consistency and stability. The main drawback is that the orientation of the ligand cannot be controlled because the coupling site cannot be specified. Another drawback is that chemical modification of the ligand could in some cases affect the analyte-ligand binding. If these issues become apparent, you can opt for the capture coupling method.
Covalent coupling of ligand to Amine sensor chips using EDC/NHS activation.
Capture coupling occurs when the ligand is immobilized through a non-covalent interaction with an intermediate capture molecule which is typically covalently coupled to the surface.
The nature of this method brings a number of advantages:
- No ligand modification required in some cases
- Ligand orientation is uniform and homogenous
- Capture molecule is specific to the ligand
Reuse and regenerate same sensor surface
4. Biotin/Streptavidin – Soul mates
Biotin and streptavidin are inseparable. They are an excellent method of immobilization because biotin and streptavidin are famous for having one of the highest affinities of any biomolecular interaction. Benefits include no background off rate to be concerned with. The interaction can even withstand regeneration conditions which resembles the nature of covalent capture.
Capture coupling of ligand to Streptavidin and Biotin sensor chips.
5. NTA Sensor Chips – His. Tagged. Ligands.
Nicoya also manufactures sensor chips coated in nitrilotriacetic acid (NTA) for capture of his-tagged ligands. It is common to use poly-histidine tags for purification purposes, which is why it can be a convenient method of immobilization as well. An advantage to NTA sensor chips is that the surface can be readily used with the easy removal of his-tagged ligand using chelating agents like EDTA.
Capture coupling of ligand with His-tag on NTA sensor chip.
6. Antibody Capture – Perfectionist
High affinity antibodies are available for many proteins and can be used for specific ligand capture. The antibody coupling method does not affect the ligand activity and allows for specific ligand orientation. However, keep in mind that regeneration will not remove the antibody from the surface either. You can also use this method to capture ligands from complex samples effectively.
Capture coupling of ligand onto Antibody-coated sensor chip.
Alright, congratulations! You now have everything you need to know to choose your sensor surface chemistry. If you’re still stuck, don’t fret. Our experts are ready to answer any of your questions or concerns.