Micropatterning of Substrates and Cells - Introduction to BioMEMS - page 51

group but share with it the potential for accepting hydrogen bonds as well as the
solvation and chain lexibility characteristics; this functional similarity between
tri(propylene sulfoxide) and oligo(ethylene glycol) groups suggests a possible gen-
eral principle for protein physisorption. By linking RGD peptides to the end-group
of a PEGylated SAM, physisorption of ECM proteins can be blocked and at the
same time the precise surface concentration of adhesive ligands needed for cell
adhesion can be tailored. PEGylated SAMs, introduced by George Whitesides' lab,
have become very popular in BioMEMS because, being alkanethiols, they can be
easily patterned by microstamping (see Section 1.6.3). However, thiols desorb with
time under water, which raises concerns for long-term studies, and require the
deposition of a gold layer, which adds cost and attenuates light for microscopic
inspection.
2.2.3 Cross-Linkers
he majority of immobilization methods used in biopatterning require not only a functional-
ized SAM but also a “ cross-linker ”—a molecule that covalently bridges two molecules, in this
case, the biomolecule of interest to the end-group of the SAM. Generally used for protein con-
jugation, most of these cross-linkers are now commercially available from a variety of sources.
Cross-linkers may have two diferent reactive ends (“ heterobifunctional cross-linkers ”) or the
same functional group at both ends (“ homobifunctional cross-linkers ”). For our purposes,
here, we only mention the few that appear most oten in the text. Glutaraldehyde is a com-
mon, inexpensive homobifunctional cross-linker that links amino groups by reaction with its
aldehyde groups. For example, an aminosilane SAM that is exposed to a glutaraldehyde aque-
ous solution readily immobilizes glutaraldehyde, thus presenting a second aldehyde group for
subsequent immobilization of a molecule which contains amino groups—such as a protein.
Other groups that are highly speciic toward amines and present in many heterobifunctional
cross-linkers are succinimidyl esters (e.g., N -hydroxysuccinimide ester —always abbreviated
as NHS ester ), isothiocyanates, and sulfonyl chlorides. hese amine-reactive groups conju-
gate with aliphatic nonprotonated amines, hence the reaction is faster at slightly basic pH.
Maleimido functionalities, on the other hand, react speciically with thiol groups—not with
amino groups. Maleimidoacetic acid NHS ester is thus a cross-linker that links amino groups to
thiol groups. A cross-linker may also contain a photoreactive group that reacts nonspeciically
upon exposure to light of a certain wavelength. Albeit expensive, photoreactive cross-linkers
have great potential for biopatterning because the reaction can be light-addressed through
a standard chrome mask or with a focused laser. Benzophenones , azides, or diazirines are
examples of photosensitive groups oten present in commercially available cross-linkers. For
instance, the amine-reactive cross-linker 4-azidosalicylic acid NHS ester contains an azide
group; the thiol-reactive cross-linker 4-( N -maleimido)benzophenone contains a benzophe-
none group. Benzophenones have the advantage, over azides and diazirines, of being chemi-
cally more stable, insensitive to ambient light or moisture, reactive with unreactive C-H bonds
even in the presence of solvent water, and activatable at wavelengths (350-360 nm) that are not
damaging to biomolecules.
2.3 Micropatterns of SAMs
he use of SAMs in micropatterning biomolecules is appealing because the adhesiveness of the
surface is engineered at a molecular level and, at the same time, patterning is reduced to pattern-
ing the SAM. Figure 2.6 depicts the main four strategies used to pattern SAMs.