Biomedical Engineering Reference
In-Depth Information
immobilize small molecules with various functional groups, the team went fur-
ther and developed an isocynanate-mediated approach, which could capture small
molecules containing alcohols, amines, carboxylic acids, thiols, phenols, and so on.
In a recent report, the group has adopted this method successfully to immobilize more
than 15,000 compounds onto glass slides and screen their binding affinity toward 100
different proteins. The compounds come from different sources, such as commercial
compounds, natural products, and synthetic libraries from academic laboratories [39].
In recent years, many research groups have developed various site-specific
approaches to immobilize small molecules onto an array. Houseman et al., for exam-
ple, utilized Diels-Alder chemistry to immobilize peptide-cyclopentadiene conju-
gates onto a self-assembled monolayer (SAM) displaying a benzoquinone handle
[40]. The process is demonstrated in Figure 13.3.2a [38]. SAMs can minimize non-
specific absorption of proteins onto a solid surface and eliminate the blocking proce-
dure in the subsequent array screening process. The reaction between benzoquinone
and cyclopentadiene is highly specific and extremely fast. Through this immobiliza-
tion strategy, the group was able to study peptide phosphorylation with Src kinase
by various compatible techniques, such as surface plasmon resonance (SPR) and a
fluorescence method. The group subsequently extended their work to adopt a photo-
chemical approach to generating patterning substrates [40] which could be used for
biospecific interaction studies. In this approach, the gold surface was modified by
SAMs with nitroveratryloxycarbonyl (NVOC)-protected hydroquinone. Under ultra-
violet irradiation, specific regions of the gold surface could be activated to release
a free hydroquinone group. This allowed for subsequent oxidation to yield benzo-
quinone, which reacted further with diene-containing molecules. Through this tech-
nique, fluoresceine and peptides with RGD sequence were immobilized successfully
onto a gold surface. The same group also developed another complementary strat-
egy using maleimide-functionalized SAMs to anchor thiol-terminating molecules
[41,42]. They immobilized carbohydrate-thiol conjugates onto a gold surface and
studied the lectin-binding profiles with SPR and fluorescent techniques. The results
of the research demonstrated that lectin could recognize the corresponding carbohy-
drate ligands on the chip.
Native chemical reaction is well-known for its chemoselectivity and bioorthog-
onal activity. It has been used by Yao's group to investigate the phosphorylation
of peptides by Src kinase on microarray (Figure 13.4b) [34,43]. As a proof-of-
concept experiment, the group first immobilized peptides with N-terminal cysteine
onto thioester-functionalized glass slides and probed the binding specificity with
their corresponding fluorescently labeled antibodies. The method was found highly
specific and efficient. Only the terminal cysteine of the peptides would react with the
thioester group on the glass surface when other internal cysteines and nucleophiles
were present. Driven by these positive results, the group delved further to study the
phosphorylation pattern of a peptide library against a kinase. Using putative p60c-
src substrate as the template, various peptides were generated via different peptide
library synthetic strategies, including alanine-, deletion-, and positional-scanning
peptide libraries. These N-terminal cysteine-containing peptides were then spotted
onto PEGylated thioester slides and used to profile the phosphorylation pattern of
