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Chapter 5
5.4.6 Microcontact Printing of PNA.
PDMS stamps were first oxidized in UV/plasma reactor (Ultra-Violet Products, modelPR-
100) for 30 min at a distance of about 2 cm from the plasma source. This reactor contains a
low-pressure mercury UV light operating with UV emissions at 185 nm (1.5 mW cm -2 ) and
254 nm (15 mW cm -2 ). The stamps thus obtained were stored in MilliQ water. Subsequently
the hydrophilic stamps were dried with nitrogen and incubated with a drop of 10 µM PNA
solution in acetonitrile for 5 minutes at room temperature. The stamp was dried with nitrogen
and brought into conformal contact with the aldehyde-terminated glass slide for 15 min using
a 15 gr weight. After printing the glass slides were immersed in a solution containing 100 mg
NaBH 4 in 40 mL PBS with 10 mL EtOH for 5 min. After reaction time the substrate was
rinsed in MilliQ water, sonicated in a carbonate buffer 0.1 M pH=9 with SDS 2% for 2
minutes, rinsed in a 1 mM carbonate buffer and dried with nitrogen.
5.4.7 Hybridization on the Substrate Surface.
For hybridization oligonucleotides were diluted to 1 µM in 4X SSC containing 0.1% SDS and
applied to the surface of the modified glass using disposable hybridization chamber (Gene
Frame, Thermo Scientific, UK). The temperature was tuned depending on the affinity of the
probe for the complementary sequence, and the time was 4 hours. Non hybridized molecules
were removed washing with SSC 2X, SDS 0.1% at hybridization temperature for 5 min. and
with SSC 0.2X at room temperature for 1 min. After washing, the glass slides were dried with
nitrogen
5.4.8 Fluorescence Microscopy
Fluorescence microscope images were taken using an Olympus inverted research microscope
IX71equipped with a mercury burner U-RFL-T as light source and a digital Olympus DR70
camera. For image acquisition blue excitation (450nm ≤ λex ≤ 480 nm), green emission
(λem≥ 515nm), green excitation (510nm ≤ λex ≤550nm) and red emission light (λem≥
590nm) were filtered using a U-MWB Olympus filter cube. All fluorescence microscopy
images were acquired in air and analyzed on a fluorescence microscope (Olympus inverted
research microscope IX71).
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