Biomedical Engineering Reference
In-Depth Information
EGF-His could trap cells as efficiently as EGF-His immobilized by surface anchor-
ing through coordination.
To prepare a Ni
2+
-chelated surface, a thin gold layer was deposited onto a glass
plate; then, on the gold surface, a SAM was formed that terminated with trivalent
carboxylic acids; finally, Ni
2+
ions were chelated to the acidic termini. In detail, first,
thin chromium and gold layers were deposited onto the surface of a glass plate with
a vacuum evaporator. The glass plate was then immersed in ethanol that contained
16-mercapto-1-hexadecanoic acid (COOH-thiol) and (1-mercaptoundec-11-yl)
triethylene glycol (TEG-thiol) at various compositions to allow the formation of
mixed SAMs. Each glass plate coated with a mixed SAM was immersed in a solution
containing
N
,
N
0
-dicyclohexylcarbodiimide (DCC) and
N
-hydroxysuccinimide
(NHS), which converted the terminal carboxylic acid to an active ester. Subsequently,
a solution of
N
-(5-amino-1-carboxypentyl) iminodiacetic acid (NTA) was plated onto
the activated surface to introduce triacetic acid. The glass plate was then immersed in
aNiSO
4
solution to form the Ni
2+
chelate. Finally, an EGF-His solution was plated
onto the Ni
2+
-chelated surface to allow immobilization of EGF-His through the coor-
dination of Ni
2+
with His. A His-tagged protein firmly binds to a Ni
2+
-chelated
substrate; for instance, His-tagged green fluorescent protein (GFP) bound to NTA-
Ni
2+
with a dissociation constant of 4.2
10
7
M[
93
]. The mixed SAM surface was
characterized by water contact angle measurements, infrared reflection adsorption
spectroscopy, and XPS. These revealed the formation of well-defined surfaces. The
expected structure of an EGF-His-immobilized surface is schematically depicted in
Fig.
6
. The surface density of immobilized EGF-His increased with increases in the
COOH-thiol content of the COOH-thiol/TEG-thiol mixture used to prepare the
SAM. The maximum EGF-His density was approximately 0.4
m
g/cm
2
with 100%
COOH-thiol.
The incorporation of TEG-thiol onto a COOH-thiol SAM elevated the fraction
of correctly oriented EGF-His on the surface by preventing nonspecific adsorption
of EGF onto the SAM surface. This might be explained by the following findings.
The area occupied by a single EGF molecule (2.98 nm
2
/molecule) [
94
] is approxi-
mately ten times larger than the area occupied by COOH-thiol (0.25 nm
2
/molecule)
[
95
] or TEG-thiol (0.35 nm
2
/molecule) [
96
]. Therefore, on a surface of 100%
COOH-thiol, most COOH-thiol molecules would not be expected to be involved
in the coordination with EGF-His. Instead, the excess carboxylic acids contained in
COOH-thiol and NTA would be expected to trigger nonspecific adsorption of EGF-
His. Under conditions where the COOH-thiol content was 10-15%, and 80% of
COOH-thiol was converted to Ni
2+
chelate, the predicted density of Ni
2+
chelate
would be equivalent to that of closely packed EGF-His in a monolayer. However,
higher surface densities of COOH-thiol gave rise to larger amounts of immobilized
EGF-His, without obvious saturation at 10-15% COOH-thiol. This was probably
due to the nonspecific EGF-His binding to residual carboxylic acids present on the
surface after NTA derivatization and Ni
2+
chelation. Furthermore, these nonspecif-
ically bound EGF-His molecules may hinder access to the Ni
2+
sites for specific
coordination.
