View - DSpace UniPR

PNA Microcontact Printing
Figure 5-6: UV-vis absorption spectra of aldehyde (red) and PNA (blue)monolayers obtained
derivatizing quartz slides
5.2.3 PNA-DNA binding properties on surface
The recognition properties of the PNAs linked to the surface were then throughly studied, in
order to determine their affinity for the complementary DNA sequences and the specificity of
the recognition. The possibility to obtain well defined patterns in the range of micrometers
and to image them with microscopy was applied to study the hybridization properties of the
PNA onto the surface, studying the affinity and the selectivity of the binding by evaluating the
melting curve of the duplex formed with fluorescent DNA oligonucleotides on surface.
Patterns of non labelled PNAs were prepared by printing PNA 2 (containing a 2D-Arginine
modified monomer) on glass slides, and hybridizing it with the complementary and
mismatched oligonucleotides labeled with TAMRA (DNA c2 and c1) at room temperature for
90 min, followed by a short wash with SSC 2x solution. In order to simulate the conditions in
which hybridization usually takes place in solution, the slides were immersed in 1 ml solution
of DNA 10 nM in SSC 2x SDS 0.1% pH=7 buffer and imaged using a laser scanner confocal
microscope during a temperature increase from 20°C to 40°C. The reason for using a confocal
microscope lies in the necessity to have a high resolution, which is necessary in order to
separate the fluorescence contribution of the solution from that of the pattern. The pictures
obtained showed a very intense and well defined fluorescent pattern at room temperature,
whose intensity and definition decreased by increasing the temperature of the solution. At the
end of the experiment the pattern was hardly detectable (Figure 5-7).
99