DISSERTATION

______________________________________________________________________ Introduction
1.3 DNA immobilization
1.3.1 DNA immobilization approaches
A DNA immobilization strategy is determined by the substrate material used for the attachment.
Over the years, various surfaces were investigated for immobilization of DNA such as among
others the hanging mercury drop electrode, carbonaceous materials, boron-doped diamond,
silver, platinum and gold. Initially, research on DNA was conducted solely on mercury drop
electrodes (in the beginning of the 1970s) and carbon electrodes (since the middle of the
1970s 46 ). Later, gold electrodes became popular, as the chemisorption of thiol-tethered DNA
showed to be a very promising method for the preparation of DNA sensors.
With respect to the type of bond formed during immobilization, DNA immobilization methods
are characterized by three main mechanisms 47 : physisorption, covalent immobilization and
chemisorption.
Physisorption is the simplest immobilization method, which is based on the adsorption of
unmodified oligonucleotides on an electrode through electrostatic forces, van der Waals
interactions, hydrogen bonds and hydrophobic interactions. The immobilization occurs either
through nucleic bases (immobilization of ssDNA, Figure 1.9, a) or the phosphate backbone
(dsDNA, Figure 1.9, b). It is characterized by a multiple site attachment, which allows for the
investigation of direct DNA oxidation and reduction. The main drawback of this method is its
sensitivity on environmental changes (pH value, temperature, ionic strength) due to the weak
attachment. Furthermore, attachment of the DNA occurs via multiple points, which prevents
further hybridization due to the restricted configurational freedom of physisorbed DNA 48 .
Carbonaceous materials and the mercury drop electrode were mostly utilized for this
immobilization technique.
Covalent immobilization results in a much stronger binding between the surface and DNA
(Figure 1.9, c). Another advantage of this method is the appropriate orientation of the probe
DNA due to the end-point attachment of ssDNA, which facilitates hybridization. In order for
the immobilization to occur, the immobilization surface (chemically or electrochemically) and
the DNA itself need to be activated, which presents a drawback of this method. Activation of
1.3 DNA immobilization 16