Biology Reference
In-Depth Information
[120]. Thiol-based linkage to the DNA means that at high coverages
the DNA is oriented normal to the electrode. If we approximate
the oligonucleotide molecule to a cylinder, then the maximum
possible loading is defined by the diameter of the cylinder. This
value is 20 A [121]. The coverage from such immobilization can be
calculated using the chronocoulometric method described by Steel
et al.
[121]. The principle of the method is that [Ru(NH
3
)
6
]
3
+
is
used to compensate the negatively charged phosphate groups of
the DNA under conditions of low supporting electrolyte. Therefore,
whenDNAisimmobilizedattheelectrodesurfacetheconcentration
of [Ru(NH
3
)
6
]
3
+
is increased. The coverage
Γ
of DNA-bound
[Ru(NH
3
)
6
]
3
+
is determined by stepping the potential to a value
where [Ru(NH
3
)
6
]
3
+
is reduced at a diffusion-limited rate. From the
integrate
d
form of the Cottrell equation [46], a plot of charge
Q
against
√
t
will have a
y
-intercept equal to
Q
dl
+
nFA
Γ
,where
Q
dl
isthedouble-layercharge,determinedfromthesamepotentialstep
in the absence of [Ru(NH
3
)
6
]
3
+
. There are
Γ
N
A
molecules/cm
2
of
[Ru(NH
3
)
6
]
3
+
ontheelectrode,where
N
A
isAvagadro'snumber,and
therefore, assuming each [Ru(NH
3
)
6
]
3
+
molecule is compensated
by three phosphate groups, and there are
m
phosphate groups
on one DNA probe, there are (3/
m
)
Γ
N
A
DNA probes/cm
2
on the
electrode. The technique can be applied to ss and dsDNA and
thus the hybridization e
ciency can be determined. Some studies
have suggested that the e
ciency decreases with DNA coverage
[122]. When thiol-modified DNA is immobilized on gold, a “diluent”
alkanethiol is often also adsorbed to displace weakly bound DNA
bases. In these cases, the chronocoulometric method has indicated
that hybridization e
ciency increases with DNA length above the
diluent layer [123].
A much simpler method of immobilization is direct adsorption,
in which case we would expect the DNA to be oriented horizontally
along the electrode. Therefore, the maximum coverage will be
determined by the number of layers it is possible to deposit.
Forces such as hydrogen bonding, base stacking, van der Waals and
hydrophobic interactions are expected to be involved [124]. Due to
thenegativechargesofthephosphategroups,theadsorptioncanbe
assisted by electrostatic attraction. Glassy carbon electrodes have
been used to bind DNA after modification by the cationic polymer
chitosan [120]. Another way to assist electrostatic attraction is to
