Hybridization of binary monolayers of single stranded oligonucleotides and short blocking molecules

We have studied the immobilization of single stranded (ss) DNA oligonucleotides of 16–27 base pairs on gold. The oligonucleotides were thiol-modified (SH-ssDNA) or disulfide-modified via a dimethoxytrityl-group (DMT-S-S-ssDNA). Immobilization was performed by adsorption of the probes on the gold surface for 10–15 min, a time within which saturation coverage was obtained for both thiol- and disulfide-modified probes. Hereafter the layer was post-treated with hydroxyalkyl substituted lipoamides also for a time of 10–15 min. The surface density of layers with shorter probes (16–18 mer) was twice (2.4 ± 0.2 × 1013 probes/cm2) that of the longer probes (25–27 mer) as studied with surface plasmon resonance. Hybridization of single stranded polymerase chain reaction (PCR) amplified products with a length above 300 base pairs gave a very low hybridization response. For amplicons with about 100 base pairs the response was high. The surface coverage was comparable to that of complementary ssDNA binding (3.0 × 1012 strands/cm2). Surfaces made from SH-ssDNA showed a 30% higher hybridization response than surfaces made from DMT-S-S-ssDNA. The PCR amplified products used are of relevance in breast cancer diagnosis. The results clearly demonstrate that the single stranded PCR products might be used in label-free cancer diagnostics.