DISSERTATION

_____________________________________________________________ Results and Discussion
with Fc-tDNA to indirectly determine the ssDNA coverage which is possible due to a low
ssDNA coverage, Figure 3.53, c. After subsequent dehybridization (Figure 3.53, d), free ssDNA
was again hybridized with a fully complementary non-labelled tDNA sequence (Figure 3.53, e)
and subsequent intercalation of AO-GOx was performed (Figure 3.53, f) followed by chronoamperometric
measurement. Afterwards, tDNA and AO-GOx were removed from the electrode
by H2O and ethanol (Figure 3.53, g), the remaining ssDNA/MCU-modified surface was again
immersed into the AO-GOx solution (Figure 3.53, h) and the signal of the negative control was
measured by subsequent amperometric experiments.
Control of the ssDNA coverage and minimization of unspecific adsorption was achieved by
careful surface preparation based on the developed potential-assisted surface modification
method. The envisaged pDNA coverage is lower than in the case of a direct detection of
hybridization (e.g., via detection of Fc-tDNA). Due to the large dimension of the enzyme
(diameter of the native enzyme is around 7 nm 108,109 ) and with this the entire intercalation
compound, ssDNA coverage must be low to prevent steric hindrance of neighboring DNA
strands towards intercalation. Shortening the immobilization time to obtain ssDNA coverage in
the order of 10 11 molecules/cm 2 provides enough space for the intercalating compound to
interact with dsDNA.
After the immobilization of ssDNA the electrode was passivated with MCU by applying the
potential-assisted method. One of the main challenges in DNA detection schemes involving
intercalators is to reduce the signal of the negative control, i.e. the interaction of the intercalator
with the ssDNA/thiol-modified surface. This interaction can occur either with the ssDNA or
from unspecific adsorption of the intercalator on the electrode surface. Since it is reported that
acridine orange solely intercalates into dsDNA 105,106 interaction with the ssDNA is expected to
be minimal. Thus, it is important to provide well-passivated surfaces that prevent significant
unspecific adsorption of the intercalator to allow for a high signal-to-noise ratio. DNA detection
via GOx amplification is based on the re-oxidation of the redox mediator at the electrode
surface. Therefore, the electron transfer between the mediator and the electrode needs to be
allowed. Previously it was reported that MCU provides the best compromise between electrode
passivation and permeability 100 . Applying the developed approach for potential-assisted
passivation of the surface with MCU for 1 min it was possible to clearly differentiate between
dsDNA and ssDNA-modified electrodes and obtain a high signal-to-noise ratio.
3.5 Intercalation as a DNA detection technique 100