W. Richard Bowen and Nidal Hilal 4
W. Richard Bowen and Nidal Hilal 4
W. Richard Bowen and Nidal Hilal 4
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7.2 ENgINEERINg THE ECM FOR PRObINg CELL SENSINg 201<br />
which react with the substrate. Upon SAM formation, the chemical properties<br />
of the surface are no longer determined by the underlying substrate<br />
but by the exposed functional tail groups at the non-substrate end of the<br />
SAM. A commonly used system is the self-assembled monolayer of an<br />
alkanethiol (SH(CH 2) nX) on gold; the sulfhydryl head groups (SH–) spontaneously<br />
form sulphur–gold bonds <strong>and</strong> leave the functional tail group<br />
structures (X) tethered away from the surface. The terminal X groups can<br />
be tailored to have different functionalities [20], e.g., NH 2– <strong>and</strong> COOH–<br />
groups for subsequent attachment of proteins or peptides via common<br />
bioconjugation chemistries [21]. The use of mixed or diluted monolayers<br />
<strong>and</strong> tuning the length of the hydrocarbon spacer chain can also be used to<br />
achieve desired surface properties [22]. SAMs of alkylsilane on hydroxylated<br />
surfaces of SiO 2/Si substrates, such as glass <strong>and</strong> silica, have also been<br />
extensively developed, although here, greater care needs to be taken over<br />
the reaction conditions to avoid self-polymerisation on the surface.<br />
As photolithography requires clean room facilities to prevent dust particles<br />
spoiling the pattern transfer process, the infrastructure costs mean that<br />
these techniques are often not easily accessible on a regular basis. Thus, a<br />
pioneering method has been developed by Whitesides’ group, called ‘soft<br />
lithography’ (microcontact printing, Figure 7.2(B)). This uses a physical<br />
stamp made from poly(dimethysiloxne) (PDMS) elastomer [23]. The stamp<br />
is a negative replica of a microstructured substrate (master) made using<br />
conventional photolithography <strong>and</strong>/or etching methods. Generally, the<br />
stamp is fabricated by thermal curing of PDMS against a master template<br />
followed by peeling away the cured structure. The PDMS stamps can then<br />
be inked with silanes, alkanethiols or ECM proteins to produce patterns<br />
on a wide range of substrates [24]. Unstamped regions can be blocked to<br />
resist non-specific protein adsorption by various methods such as the use<br />
of PEG-terminated SAMs or rinsing with denatured albumin solution. The<br />
PDMS stamps can be repeatedly replicated from the master <strong>and</strong> reused<br />
many times, thereby significantly reducing the cost of fabrication. Using<br />
this methodology, microcontact printing has greatly extended the range of<br />
substrates that can be engineered for use in cell studies since many of the<br />
polymeric materials compatible with biological studies are not suitable for<br />
patterning using traditional photolithography processes.<br />
An early example of the use of micropatterned surfaces is one which<br />
allowed systematic examination of the interaction of cells with substrate<br />
surface chemistries <strong>and</strong> ECM components [25]. Observations of cell<br />
growth on metallic <strong>and</strong> polymeric micropatterns have provided invaluable<br />
information to develop new types of cell culture vessels <strong>and</strong> screen<br />
the biocompatiblity of metal materials for implants. Cell function data on<br />
SAM patterns have enabled the identification of chemistries <strong>and</strong> systems<br />
that possess designed properties; e.g. observations from patterns made<br />
using a range of poly(ethylene glycol) (PEG)-derivatised alkanethiols