Biomedical Engineering Reference
In-Depth Information
Another well-known noncovalent interaction pair is streptavidin and biotin. The
affinity between streptavidin and biotin is very strong. It can resist various harsh
conditions such as extreme pH, organic solvent, and heat. It has been used widely for
biochemistry studies, including protein enrichment and protein-protein interaction
assays. In 2002, Lesaicherre et al. applied this technique successfully to immobilize
biotinylated peptides onto avidin-functionalized glass slides (Figure 13.3b) [34]. The
peptide array generated was used to study specific interactions between peptides and
antibodies. Apart from providing immobilization functions, avidin can also serve as
a molecular layer. It effectively blocks nonspecific binding of proteins with the slide
surface. The approach was subsequently applied by Uttamchandani and Yao in a
series of substrate fingerprinting studies of various proteins [35].
Fluorous-fluorous interaction has been exploited by several groups to probe non-
covalent immobilization of small molecules onto an array (Figure 13.3c). Ko et al.
developed a fluorous-based approach by immobilizing carbohydrates with a C
8
F
17
tail onto a fluorinated glass slide [36]. The group demonstrated that the interaction
between the C
8
F
17
tail and the fluorinated surface is robust enough to withstand strin-
gent wash conditions. After immobilization onto an array, the carbohydrates could
recognize a fluorescently labeled lectin specifically. Recently, Vegas et al. constructed
a fluorous-based SMM to screen histone deacetylase (HDAC) inhibitors [37]. Twenty
fluorous-tagged small molecules were printed onto an array and screened against
HDAC2, HDAC8, and HDAC3/NCoR2. The method produced an excellent signal-
to-noise ratio and proved its potential as an effective tool in identifying selective
HDAC inhibitors. In a similar approach, Nicholson et al. proved that a fluorous-based
microarray was capable of detecting protein-ligand binding events [38].
13.3.2 Covalent Immobilization Approach
Unlike noncovalent interactions, covalent immobilization generates a firm and stable
linkage between small molecules and an array surface. For example, small molecules
containing primary amines could be firmly immobilized onto epoxy-, aldehyde-, and
NHS-coated slides. Depending on whether the immobilization tag is unique, covalent
immobilization methods can be further divided into random immobilization and site-
specific immobilization. If there is more than one immobilization tag present in the
of molecules, it will lead to random immobilization. As a result, the immobilized
molecules could possess multiple orientations in the same spot. This will lead further
to inhomogeneous molecular interactions. Introducing a unique tag during library
synthesis is often tedious. Nevertheless, the time and effort spent pay off, as they
ensure that the immobilizedmolecules display uniformorientation, thereby enhancing
molecular interaction.
The Schreiber group has developed several array immobilization approaches to
anchor small molecules derived from a DOS library. The group first fabricated a chlo-
rinated glass slide to catch DOS-derived compounds containing a hydroxyl group.
Following that, they developed a strategy to coat the glass surface with a diazoben-
zyldiene group, which could catch phenol and other acidic functional groups. To
