Biomedical Engineering Reference
In-Depth Information
a tyrosine kinase, p60c-src.The results of the experiment indicated clearly that a
peptide array can serve as a competent approach to assaying kinase activity.
Besides native chemical reactions, many other chemoselective reactions have been
explored for small molecule immobilization. Falsey et al. prepared a new type of
microchip by installing a glyoxylyl group on the slide surface [44]. The authors
successfully anchored aminooxy- and cysteine-containing small molecules onto
glyoxylyl-derived glass slides with two different chemical reactions. They employed
this method in a protein-binding study, a kinase assay, and a cell adhesion study.
Using a similar process, Melnyk et al. synthesized glyoxylyl-linked peptides and
immobilized these peptides onto semicarbazide-derivatized glass slides [45]. The
group showed that the peptide array constructed was highly specific and sensitive to
the antibody tested. Kohn et al. and Soellner et al., independently, developed methods
to anchor azide-containing small molecules onto phosphine-derivatized slides using
Staudinger ligation (Figure 13.4c) [46,47]. It is noteworthy that Staudinger ligation
is very mild and efficient even in the presence of water and oxygen.
In 2004, the Wong group functionalized the surface of microtiter plates with a
cleavable alkyne handle and introduced azide-containing oligosaccharides onto the
solid surface (Figure 13.4d) [48]. Through the addition of Cu(I), click chemistry
could occur efficiently between an alkyne and an azide in aqueous solution. The
group demonstrated that this approach could be used for lectin/antibody binding
studies. Additionally, a cleavable linker allows easy characterization of carbohy-
drates and quantitative analysis with MS analysis after DTT cleavage. Although this
immobilization chemistry was done with microtiter plates, the method can readily
be applied to glass slides. Through thiol-ene click chemistry, Jonkheijm et al. also
used a new photochemical method for surface patterning [49]. The approach can
be used to immobilize olefin-containing proteins/peptides onto a thiol-derivatized
surface through irradiation. The immobilization reaction was complete within 10
min, and the dimension of surface patterning could reach the submicrometer range.
In 2005, Lee and Shin employed a chemoselective immobilization approach to link
hydrazide substrates covalently to epoxide-coated slides [50]. No cross-reaction was
observed in the presence of amines and hydroxyl groups, and the method was shown
to be highly selective. It was also noted that strong nucleophiles such as thiols would
undergo a slow reaction with epoxide-derivatized surface at low pH values, whereas
hydrazide could react with epoxide efficiently at pH 3 to 5.
The aforementioned approaches share the fact that they all require a unique tag
in the presence of molecules. This is to allow for the site-specific attachment of
molecules onto the solid surface. Introducing a special tag into molecules can be
realized through combinatorial library synthesis. However, some small-molecule
libraries without any tag or reactive functional groups pose difficulty to SMM screen-
ing. It is especially evident for natural product libraries derived from natural sources.
To overcome this problem, Kanoh et al. developed a random covalent immobiliza-
tion approach through a photoaffinity reaction [51]. They modified the glass surface
with a photoaffinity linker containing diazirine. The surface underwent irradiation to
activate the diazirine group and release the carbine species. The carbine species were
then inserted in the neighboring molecules to form strong covalent bonds. Various
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