Characterization of PNA and DNA immobilization and subsequent hybridization with DNA using acoustic-shear-wave attenuation measurements

We report here how the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, simultaneously measuring changes in the induced energy dissipation, D (c.f. viscoelastic properties), and the frequency, f (c.f. coupled mass), can be used to characterize the bound state of single-stranded peptide nucleic acid (PNA) and DNA in relation to their ability to function as selective probe(s) for fully complementary and single mismatch DNA. The possibility to use the QCM-D technique for detection of binding kinetics and structural differences in the formed duplexes is also presented.

We found that thiol-PNA and thiol-DNA attached via a sulfur group directly on a bare gold surface are less efficient as probes for DNA than are biotin-PNA and biotin-DNA, coupled on top of a two-dimensional (2-D) arrangement of streptavidin, formed on a biotinylated phospholipid bilayer on a SiO₂ surface. The fully complementary and singly mismatched DNA oligomers hybridize with the immobilized PNA and DNA. A single mismatch is discriminated via a significant difference in the binding and dissociation kinetics, demonstrating a high selectivity and thus successful immobilization of functional single strands. The observed ratios between hybridization-induced energy dissipation (DD) and the frequency shift (Df) allowed to discriminate thiol-PNA directly attached to a gold surface from biotin-PNA coupled to the streptavidin-2D-arrangement, where the former were shown inefficient for detecting subsequent hybridization. Structural differences of the immobilized layers composed of biotin-PNA-DNA and biotin-DNA-DNA was clearly reflected by the DD and Df response.