Covalent immobilization via EDC/NHS Advantages: + Well described standard method + Zero-length linker + High immobilization capacity Disadvantages: - Non-physiological conditions for the ligand in immobilization buffer - Neutralization of ligand lysines can result in unpredictable activity losses - Immobilization buffer scouting is mandatory for new ligands - Difficult to target a precise immobilization level - Rapid hydrolysis of activated carboxylic groups - Almost no ability to add ligand to achieve a higher immobilization level - Risk of ligand and hydrogel crosslinking - Non-selective and difficult to control side reactions possible - Quenching step required - Poor reproducibility of immobilization levels
Immobilization via biotin/streptavidin Advantages: + Fast and easy – established system + Physiological conditions during labeling and immobilization + Possibility to target a precise immobilization level + No quenching step required + Possible to re-immobilize ligand to aim for a higher immobilization level + No crosslinking, if biotin/ligand ratio < 1 Disadvantages: - Pre-immobilized streptavidin occupies a significant portion of the available space in the evanescent field - Higher non-specific binding (NSB) of analyte compared with sensor chips without pre-immobilized streptavidin - Generally lower ligand density compared to other methods - Diffusion artefacts through steric hindrance of bulky streptavidin linkers
Immobilization via Ni-NTA Advantages: + Oriented ligand immobilization + No crosslinking + Reversibility of immobilization if ligand cannot be regenerated or loses activity when exposed to the analyte + No quenching step required + Small linker Disadvantages: - Significant non-specific binding of sample matrix and analyte possible – especially with larger proteins - Relatively low immobilization capacity - Unstable immobilization in buffers containing mM concentrations of DTT and/or EDTA
Immobilization via Click-Coupling Advantages: + Small linker + Extremely low non-specific binding of the azide activated chip surface + In many cases drastically increased ligand activity + Precise targeting of immobilization level + Highly selective chemical coupling + Oriented immobilization possible, if ligand contains azide modified amino acids + No quenching step required + High immobilization capacity + Immobilization at physiological conditions possible + No cross-linking if label/ligand ratio < 1 + Pre-activated and long-term stable sensor chip surface + Ability to re-immobilize ligand in significant amounts to achieve higher immobilization level + Very high chip to chip reproducibility Disadvantages: - Somewhat longer contact time of the chip surface / ligand required, especially at physiological conditions