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PNA Microcontact Printing
5.4.9 Surface Melting experiments.
The samples for the melting experiments were prepared printing the PNA, hybridizing it with
a 1 µM solution of TAMRA labeled oligonucleotide and washing with SSC 2X at room
temperature. The samples, thus prepared were immersed in a 10 nM solution of the same
oligonucleotide in SSC 2X SDS 0.1% buffer and imaged using a laser scanning confocal
microscope during temperature increase the temperature.
5.4.10 Laser scanning confocal microscopy
Confocal microscopy images of the microcontact-printed substrates were taken on a Carl
Zeiss LSM 510 microscope. The light was focused on the substrate using a 40X LD Acroplan
objective. TAMRA derivatised features were excited at 543 nm, using a Helium-Neon laser.
The emitted fluorescence was collected on. The images were acquired in a 10nM solution of
TAMRA labeled oligonucleotides in a SSC (2x) SDS (0.1 %) buffer pH= 7. Temperature was
regulated using a Temperature Controller TC202A Harvard Apparatus with a heating stage.
Real temperature was checked using a thermocouple dipped in the solution. Fluorescence
intensity was calculated in three different areas of the image and averaged; the value was
normalized subtracting the background intensity calculated in the same way.
5.4.11 Contact Printing by Robotic Systems.
The spots of oligonucleotides were fabricated using Scienion S3 spotter (Scienion AG, Berlin,
GER). Spots of 350 pL were dispensed using the piezo non-contact printing system in a 16x8
array. Flat, 2-3 mm thick stamps were glued to a glass microscope glass, oxidized and used as
substrates for PNAs spotting. The PNAs used for this fabrication were prepared in water at
concentration from 1 to 50 µM. After 15 minutes from the spotting, the stamps were used for
multiple microcontact printing. Samples were hybridized as described before and scanned
using a fluorescence scanner.
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