Biomedical Engineering Reference
In-Depth Information
chain C-termini); while on some other occasions, the candidate units for
protein conjugation can also be pendant hydrosulphonyl [- SH] (of cystine
residues) or phenol (of tyrosine residues). Therefore, the bioconjugate tech-
nique for protein immobilization is actually a coupling chemistry between
these reactive groups of both sides. Water is usually the only good solvent
formostofthesolubleproteins,andconsideringthistheStepII(protein-
substrate coupling) reaction must be performed in an aqueous medium with
mild temperature, pH and salinity conditions [113-120].
The reaction Step I (substrate surface activation) and Step II (protein-
substrate coupling) can be intermediated by activating-and-leaving reagents
or multi-functional coupling reagents, which demonstrate totally different
mechanisms. The activating-and-leaving reagent behaves like a catalyst that
renders an active leaving group onto the substrate surface reducing the re-
active threshold for the following proteinic ligand substitution; while the
multi-functional coupling reagent physically tethers the ligand and sub-
strate together by covalently bonding them both, namely, playing the role
of permanent bridge. A combination of these two strategies is also widely
used. The popular activating-and-leaving reagents are
N
-hydroxy succin-
imide esters [NHS] (and water soluble sulfo-NHS), dicyclohexyl carbodiimide
[DCC] (and water soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
[EDC]),
N
,
N
-carbonyldiimidazole [CDI], sulfonyl chlorides, and 2-fluoro-
1-methylpyridinium toluene-4-sulfonate [FMP], etc. The relevant model
reactions are listed in Table 1A. The multi-functional coupling strategies
Table 1
Covalent surface immobilization of proteinic affinity ligands [124]
A. Typical activating-and-leaving strategy and reagents:
1.
N
-hydroxysuccinimide esters [NHS]
2. dicyclohexyl carbodiimide [DCC]
