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Arg-PNA Microarrays
a surface. Although methods performed in solution are usually more affordable and robust,
surface systems display several features not achievable with other techniques. The
concentration effect obtained on the surface where the probe is deposited usually results in a
sensitivity increase of the method. Surface devices are fabricated with microsystems that
allow to work with only very small amounts of probes, and small volumes of analyte solution
for the hybridization process. The possibility to use multiple probes at the same time enables
this technique to simultaneously give a large number of information and so to reduce the
number of the necessary analyses. Finally, when the system has been used, it can be recycled
by denaturating the duplex formed on the surface and thus leaving the probes linked to the
surface ready to be further rehybridized.
The possibility to freely modify the surfaces allows to exploit different physical properties for
revealing the duplex formation. The improvement of these techniques led to development of
optical, electrochemical, or mass sensitive devices. Detection methods by fluorescence are
mostly used being fast and very sensitive. In order to achieve optimal results, the probes
should be highly selective 10 . Oligonucleotide probes suffer from two drawbacks: the strong
dependence of the duplex stability upon the ionic strength 11 and the inability of the
oligonucleotide probes to invade DNA secondary structures, which disfavours the target
hybridization on the surface 12 . Therefore, these systems may be improved by substituting
oligonucleotide molecules with other similar compounds with improved specificity and
selectivity. Typical example of such compounds are Peptide Nucleic Acids (PNAs) 13 .
PNA microarrays can be made by directly linking the probes to a surface (glass or polymeric)
through a covalent linkage with suitably active functional groups. PNA microarrays can be
built by deposition of pre-synthesized and purified probes 14 , or by in-situ synthesis of PNAs 15 .
PNA probes have particularly suitable features which allow them to be applied extensively for
the analysis of particular DNA sequences 14,16 , or for the detection of SNPs 15,17,18 , obtaining
good performances. First experiments in this field were carried out hybridizing amplified and
labeled DNA samples 12 on surface and reading the fluorescence by laser scanners studied for
these systems. This method is still the most used because it is robust and reproducible.
Commercial PNA arrays exploit this detection method, albeit fluorescent labeling is not
introduced directly, but in two steps; amplified DNA is labeled with a biotin and, after
hybridization and washing are performed, a streptavidin-fluorophore is coupled and bound to
the DNA 19 . Although fluorescent detection is still the most used, many efforts have been done
for improving the detection performances, either by changing the detection method, or by
improving the probe performances, introducing ad hoc modifications. Early works were
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