DISSERTATION

_____________________________________________________________ Results and Discussion
immobilization time employed in the experiment (30 s) is not optimal and already leads to a too
high ssDNA coverage for the employed detection scheme resulting in a decreased hybridization
efficiency. By doing this the rest of the electrodes were longer exposed to immobilization by
incubation allowing to investigate, how a possible contamination influences the quality of the
chip preparation. After potential-assisted immobilization of FRIZ ssDNA and passivation with
MCU, region a was also exposed to E. coli ssDNA. There was no signal upon hybridization
with E. coli tDNA showing the high integrity of the formed FRIZ/MCU films.
Region b was exposed to FRIZ ssDNA solution, and subsequently to the MCU passivating
solution and finally to the E. coli ssDNA solution, with immobilization occurring by incubation
in all cases. Prior to the final potential-assisted passivation with MCU, the electrodes were
cleaned using the potential-assisted desorption method. Thus, this region should be modified
only with MCU. Figure 3.50, b and c confirm this by showing the absence of any signal from
FRIZ and E. coli tDNA (grey curve). In order to prove that the absence of signals is due to a
successful desorption and not due to an undetectable amount of immobilized DNA, region d
was exposed to same conditions, without performing any desorption step. That region should
be modified with both FRIZ and E. coli ssDNA sequences and passivated completely with
MCU. In both cases (Figure 3.50, b and c, beige curve) a very small amount of tDNA is detected
upon hybridization with both FRIZ and E. coli tDNA, however, this signal is negligible as
compared to the signal obtained in the regions with potential-assisted immobilization.
Nevertheless, the amount of contaminating DNA is measurable, proving that desorption in
region b was done successfully.
Finally, region c was initially exposed to FRIZ ssDNA solution at OCP, after which the
electrodes were cleaned by potential-assisted desorption and subsequently immobilized with E.
coli ssDNA and passivated with MCU by the potential-assisted method. Therefore, it is the only
region that should show a signal upon hybridization with the E. coli tDNA sequence (Figure
3.50, c, green curve). The absence of any peak upon hybridization with FRIZ tDNA (Figure
3.50, b, green curve) also indicates that desorption was performed successfully in this region as
well.
Potential-assisted methods for immobilization and desorption allow for a fully
electrochemically controlled preparation of DNA chips within a very short time. The big
advantage of the potential-assisted immobilization method is that it provides the desired DNA
coverage and blocking of the surface tremendously faster than the process at OCP and by this
3.4 Potential-assisted preparation of DNA sensors 94