DISSERTATION

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Results and Discussion
threshold 34 . Thus, DNA is screened more in solutions of higher ionic strength, decreasing by
this the ability of the DNA to repel molecules of the redox mediator. Therefore, in order to
achieve the desired sensitivity, the ionic strength of the working solution should be low enough
to allow for DNA to manifest high enough effective negative charge to significantly block the
redox mediator. On the other hand, the ionic strength needs to be high enough to not affect the
stability of the double helix during measurements with dsDNA-modified electrodes.
Figure 3.6 represents a schematic view of the electrode surface during the preparation of a DNA
sensor via a two-step immobilization method. Initially, a thiol-tethered ssDNA is immobilized
on the electrode creating a negatively charged interface (Figure 3.6, a and b). Consequently, the
approach of the redox mediator is hindered and the electron transfer rate decreases. In EIS this
is observed as an increase in Rct (Figure 3.7). The increase in Rct depends on the amount of
immobilized DNA, where a higher increase is observed for a higher ssDNA coverage. It should
be noted that the obtained EIS response is a result of the repulsion of the redox mediator by
both negatively charged immobilized ssDNA and unspecifically adsorbed DNA strands 73 , as
well as steric hindrance caused by lying ssDNA that physically blocks the access of the redox
mediator. Due to the fact that ssDNA behaves as a flexible coil and that it orientates randomly
on the electrode surface, especially at lower ssDNA coverage this response lacks in
reproducibility.
Figure 3.6. Scheme of the electrode surface during the build-up of the DNA sensor: a)
bare electrode, b) ssDNA-modified electrode, c) ssDNA/thiol-modified electrode.
Therefore, upon immobilization the electrode surface is covered with a mixture of DNA strands
that are chemisorbed via a Au-S bond and DNA strands that are bound to the surface through
the DNA backbone or bases. Additionally, grafted DNA strands also adsorb to the electrode by
3.2 Importance of knowing the surface 36