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Arg-PNA Microarrays
PNA-microarray platforms were built by using glass slides derivatised with N-
hydroxysuccinimide active groups, which were allowed to react with the PNA terminal amino
groups. The solutions for PNA spotting were prepared by dissolving the PNA probes (50 µM)
in carbonate buffer (0.1 M, pH=9) containing SDS (0.001%). The deposition of the probes on
the surface was done by using a pin system spotter.
Since PNAs are known to be strongly absorbed on many types of surfaces, three so called
“washing-spotting” cycles had to be introduced after each PNA deposition, in order to avoid
false positive signals due to the contamination of the pin. The optimization of the washing
step was done by spotting a PNA, followed by two or three spotting of washing solutions, and
finally spotting the same buffer used to spot the probe, in order to check if any residue
contamination was present in the pin. The essays allowed to evaluate the “washing power” of
different mixtures, looking for the one able to completely remove the undeposited PNA from
the pin. Washing solutions were prepared using organic solvents (such as acetonitrile and
dimethylformamide) and different mixtures of SDS (Figure 4-3). The effect of pH on the
ability to solubilize the adsorbed probe on the pin was also tested using different buffer
mixtures (Figure 4-4).
Acetate Buffer pH=4
Formiate Buffer pH=4 The results showed in
PNA Wash Wash Wash Buffer PNA Wash Wash Wash Buffer
figures 4-3 and 4-4
indicate that the best
results in terms of lack of
cross contamination is
Tris Buffer pH=9
BorateBuffer pH=9
obtained with the same
PNA Wash Wash Wash Buffer PNA Wash Wash Wash Buffer
buffer used for depositing
the PNAs, composed by
0.1 M sodium carbonate
buffer at pH= 9 with SDS
Carbonate Buffer pH=9
present at various
PNA Wash Wash Wash Buffer
concentration (0.2%, 1%
and 0.001%); after these
cycles no traces of PNAs
were detected upon
spotting (as confirmed by
Figure 4-4: Optimization of washing procedure (effect of pH and
buffer)
the lack of hybridization
with the complementary Cy5-oligonucleotide). Deposition efficiency was checked in every
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