DISSERTATION

_____________________________________________________________ Results and Discussion
Even though the employed pulse profile is within a safe potential window of the Au-S bond,
fast pulsing still provokes a partial desorption of DNA molecules, albeit at a much slower rate
than with the pulse profile employed for potential-pulse assisted desorption (0.9/-0.9 V, 10 ms).
This raises the question about why the same pulse profile results in a significantly accelerated
DNA and thiol immobilization. The reason is that the immobilization rate is apparently much
higher than the desorption rate and it prevails, leading to a very fast immobilization kinetics.
After DNA immobilization, the following passivation step should be performed within a time
window that provokes a negligible desorption of the previously formed DNA layer, which is in
this case 1-2 min. In order to find the optimal passivation procedure, potential-assisted
immobilization of MCH and MCU was performed for a duration of 1 min and the obtained
layers were compared with respect to their integrity using CV. Furthermore, the ability of
formed layers to block the unspecific adsorption of tDNA was investigated by measuring the
ferrocene signal from ferrocene labelled tDNA (Fc-tDNA) using fast scan cyclic voltammetry
(FSCV) upon incubation of modified electrodes into the Fc-tDNA solution. Since the SAM
formation of MCU is more efficient as compared with MCH in the investigated time, which is
observed as a better blocking of the redox mediator (Figure 3.46, a) and absence of unspecific
adsorption of the Fc-tDNA (Figure 3.46, b), MCU was chosen for the potential-pulse assisted
passivation step in the build-up of the DNA assay. This finding is expected since longer
alkylthiols produce a more ordered SAM with less defects 12,99 .
A crucial step in designing a DNA sensor is the optimization of the ssDNA coverage, since it
determines both the hybridization efficiency and its kinetics. To demonstrate the use of the
potential-pulse assisted immobilization method for the preparation of a DNA sensor, the ssDNA
coverage was optimized for hybridization detection based on the measurement of the ferrocene
(Fc) signal from a Fc-tDNA. Both ssDNA immobilization and subsequent MCU passivation
were performed using the potential-assisted method (0.5/-0.2 V, 10 ms pulse duration). The
ssDNA immobilization duration was varied and passivation was kept at 1 min. Sensor
preparation was characterized by CV, following each step of the build-up (an example is shown
in Figure 3.47, a). The prepared sensors were subjected to hybridization with a fully
complementary Fc-tDNA. FSCV was used for the detection of the ferrocene signal (Figure
3.47, b) and the calculation of dsDNA coverage according to equations from Section 5.11.1.
The obtained results are shown in Figure 3.48. When the ssDNA coverage on the surface is low
that adjacent DNA strands do not interfere with each other neither sterically nor
3.4 Potential-assisted preparation of DNA sensors 88