Peptide nucleic acid (PNA) is one of the most interesting and versatile artificial structural mimic of nucleic acids (NA), introduced by Nielsen in 1991.1-4
It has a neutral pseudopeptide backbone replacing the negatively charged sugar-phosphate chain of NA. The backbone is made of N
-(2-aminoethyl)glycine units linked in a polyamide structure, on which the nucleobases are attached to the α-nitrogen atom of the amino acid unit through methylene carbonyl residues. PNA features a high chemical stability, as well as a high resistance to cellular enzymes. Thanks to the presence of the nucleobases, which are at the same distance from each other as in DNA or RNA strands, PNA is able to recognize and bind in a very high specific and selective manner the cDNA and RNA, and generally it exhibits excellent mismatch sensitivity. This feature makes PNA exceptionally attractive for DNA recognition5
and, in particular, a lot of interest has been devoted to the use of PNA as the probe in different genosensors.6-8
Among the transduction mechanisms proposed, microgravimetry,9
optics, namely surface plasmon resonance technique,10,11
have been proposed. Amperometric sensors are acknowledged to be particularly effective, also easy to use and cheap; in addition, they are characterized by short response time and can be very simply miniaturized, making them portable and suitable for in vivo detections.
Many strategies have been proposed to quantify the amount of NA finally hybridized with PNA chains on the electrode surfaces. Due to the electrochemical inertness of NA chains, the amperometric signal is collected indirectly. Among the possible strategies proposed, “label free” approaches are considered to be definitely preferable, because they do not require to chemically functionalize the NA mixture under analysis. Similar approaches are effectively feasible in the frame of amperometric sensing. The amperometric transduction may take advantage of the neutral character of PNA structure, in contrast to the negative nature of DNA chains.13-19
Before the hybridization with the target NA, a negatively-charged electroactive probe in solution, such as [Fe(CN)6
, can easily interact with the electrode surface to induce the electrochemical reaction: a current signal due to its reversible oxidation to [Fe(CN)6
is registered. On the contrary, when oligonucleotide chains are bonded to the PNA on the surface, electrostatic repulsion between DNA-PNA duplexes and the redox probe occurs, giving rise to a decrement of the signal due to Fe(III)/Fe(II) redox couple. The entity of current decrement is directly related to the number of NA chains fixed on the electrode surface and, in its turn, to the concentration of the target DNA chains in the hybridization solution.
Due to all properties described, it is evident that the neutral character of PNA structure on one hand allows the formation of stable hybrids with NA, and on the other hand assures a high sensitivity in amperometric transduction, where the difference of surface charge before and after the recognition event is highly desired.15,16
For such an application, PNA chains ought to possess functional groups suitable for their stable anchoring on the electrode surface. One of the most popular groups used to such a scope is the thiol functionality,13-17,20,21
due to its high affinity for gold surfaces, in turn very frequently used as electrode substrates. However, thiol functionalized PNA results poorly soluble in water and can rapidly oxidize in this solvent.
In principle, alternative functional groups can be used to provide for stable deposition of PNA on gold surfaces. As an example, several papers report that primary amines can be fixed on gold surfaces.22-25
However, the nature of N-Au interaction has not been completely clarified and to best of our knowledge amine-functionalized PNA chains have never been used for the development of amperometric biosensors. With this respect, amine functionalities can be very easily inserted into PNA chains, also with the possible advantage to increase the solubility of this probe molecule in water, thus facilitating the grafting process on surfaces.
The possibility of synthesizing PNA modified with additional amino groups has already been reported in the literature and has some rationale. In particular, in a research aimed at solving some of the drawbacks related to the PNA use, such as a scarce water solubility, some of the present authors have designed and synthesized new PNA monomers, dimers and oligomers, named hydrazino PNA (hyd
in which the terminal amino group of the classical aeg
PNA was replaced by a hydrazine moiety. PNA decamers obtained by inserting one or more hyd
PNA monomers exhibit increased water solubility with respect to decamers containing all aeg
PNA monomers, with beneficial effect for biological studies. In another context, Marchelli et al. have shown that a PNA oligomer containing three modified monomers with chiral lysines (a chiral box in which, in addition to the stereogenic centers, three additional amino groups are present) is excellent in discriminating mismatched and matched targets; moreover, the chiral box allowed the formation of the anti-parallel PNA-DNA duplex, whereas the parallel PNA-DNA duplex failed to form.27,28
Finally, the insertion of one or more lysine residues as a tail into a PNA backbone has been reported in the literature,29
and gives some beneficial effects, for example on the solubility in aqueous solutions. More recently, Ly30
has reported an interesting miniPEG modified PNA which not only exhibits a higher water solubility but also a less aggregation propensity and a decreased tendency to randomly foil if compared with the corresponding unmodified parental PNA oligomers. Appella has also shown that the presence of l
-lysine residues in the γ-position on the aegPNA backbone not only increases the stability of duplexes with complementary antiparallel DNA, but, in addition, the presence of the lysine amine groups allows the support of a broad range of functional groups without modifying the ability of PNA to recognize complementary nucleic acids.31
In this work we have considered the use of the homothymine PNA decamer terminated with four lysine residues (aegPNA)10-lys4
1 (). In addition to increase the solubility, the role of four primary amino groups is to enhance the stability of the anchoring to the Au surfaces. The peculiar oligonucleotide sequence chosen should be considered as a benchmark recognition element for NA, devoted to study the effective use of amino terminated PNA in the frame of amperometric genosensors. This terminal group constitutes a valid alternative to the usually employed thiol functionality.
Figure 1. Molecular structure of (aegPNA)10-lys4 1 and scheme of genosensor detection strategy.
also reports schematically the whole strategy of genosensor detection. In order to improve the sensitivity of the amperometric sensor, the electrode surface consisted of a gold nanostructure, realized by stably anchoring chemically synthesized AuNPs on a gold electrode through a dithiol linker, i.e., BDT. With this respect, it is important to underline that the synthesis of AuNPs has been performed in order to make them surrounded by a particularly labile encapsulating agent, such as chloride ion, that can be easily substituted by species bearing functions with higher affinity to Au. The mechanism of chemical interaction of the amine terminal group with this kind of AuNP and with the BDT/AuNP nanostructured surface has been deeply studied through spectroscopic investigations performed with C6NH2, used as the model molecule to simulate the binding mode of the (aegPNA)10-lys4 decamer primary amino groups.