Adsorption of DNA oligomers on solid substrates has become a common practice for base sequence identification and various other applications. However, characterization of the adsorbed layer is always a problematic process that requires the application of many experimental tools. To be able to investigate self-assembled monolayers of thiolated DNA oligomers on gold substrate, we spent extensive efforts in their characterization. In addition, control experiments were performed to ensure the validity of the electron-transmission studies. In what follows, monolayer preparation and characterization as well as control experiments are described in detail.

HPLC-purified oligomers used in the experiment were checked for length homogeneity and proper thiol modification. Fig. 6 shows a PAGE analysis of radiolabeled oligos. The thiolated oligo with the linker –(CH₂)₃-S–S-(CH₂)₃ and the nonthiolated oligo (OH) have the same sequence and differ only by the disulfide linker. This difference is resolved by the gel, proving that indeed the thiolated oligo contains a modification. After addition of a reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] to the thiolated oligo, the oligo migrates faster, because the modification has been converted from a disulfide –(CH₂)₃-S–S-(CH₂)₃ to a thiol –(CH₂)₃-SH.

Thickness of the Monolayer: Spectroscopic Ellipsometry. The thickness of the monolayers was determined by applying spectroscopic ellipsometry using a multiwavelength (370-1,000 nm) ellipsometer (Woolam M2000V, Lincoln, NE) and fitted over all wavelengths with WVASE32 software. Table 2 shows the thickness measured for the different monolayers studied (see Fig. 7).

Although the thickness of the layers made from ss oligomers are all identical within the accuracy of the measurements, the layer made from the double strand (G₃C) is clearly thicker. This finding is consistent with the dsDNA being more rigid, whereas the ss oligomers are not necessarily stretched completely.

Topography: Tapping-Mode Atomic Force Microscopy. The topography of the layer was checked by atomic force microscopy (Nanoscope IIIa, Digital Instruments, Santa Barbara, CA) in tapping mode at ambient conditions (22° C, 60% relative humidity) with a DNA monolayer self-assembled on atomically flat gold slides (111) with large terraces. Fig. 8 shows the atomically flat gold substrate and the same substrate covered with various DNA layers. From both the 3D images as well as the height variations it is clear that the organization of the layer is similar for all oligomers independent of their bases. The data also indicate that the molecules are distributed evenly on the substrate.

Density of the Oligomers in the Monolayer: Radioactive Labeling [³²P]. The oligomers were radiolabeled with radioactive phosphate ([³²P]) and then self-assembled as a monolayer. The density of adsorbed molecules was quantified with a phosphorimager (Fuji). The results are shown in Table 3. The coverage is about the same (» 1.4 ´ 10¹³) for all the monolayers within the error of the radioactive measurements.

Surface Wettability: Contact-Angle Measurements. Contact-angle measurements were performed with deionized water (Millipore) by using an automated goniometer (Model-100, Rame-Hart, Mountain Lakes, NJ). The measured contact angles for different DNA monolayers are shown in Table 4.

Chemical Composition: XPS Measurements. X-ray photoelectron spectroscopy (XPS) measurements were performed by using a Kratos Analytical AXIS-HS instrument with a monochromatized Al(Ka ) source (75 W) and with pass energies within 20 and 80 eV. The results are shown in Fig. 9, and a comparison between high-resolution XPS data for two representative DNA samples differing in the number of G bases (1G and 8G) is shown in Table 5. The measured atomic ratios for both samples are nearly equal and are very similar to the calculated ratios.

Several control experiments were performed to verify the validity of the electron-transmission studies. The first experiment checked whether the UV light, used for ejecting the electrons, damages the adsorbed layer.

Fig. 10 shows gel electrophoresis analysis of radiolabeled DNA after the oligomers in solution were exposed to 193-nm light with an energy density (100 nJ/cm²) 50 times larger and 10⁶ times longer exposure time (14 sec) than that used in the experiment (2 nJ/cm² and 20 m sec). It is clear that, under these conditions, no ss breaks can be found in the DNA.

To verify that the electron produced in the electron-transmission studies were indeed produced by a single photon, the electron signal was monitored as a function of the laser intensity. The results are presented in Fig. 11 and indicate that there is a linear dependence of the electron signal on the laser flux.

To probe the effect of the salt (and counter ion) on the results, DNA monolayers on gold were prepared from an ethyl-alcohol solution instead of water, and electron-transmission experiments were performed. The results were identical to those obtained for monolayers made from aqueous solutions of DNA.

The electron-transmission spectrum for clean gold substrate, as shown in Fig. 12.