Quantitative Analysis and Characterization of DNA Immobilized on Gold

Abstract

Abstract:We describe the complementary use of X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy to quantitatively characterize the immobilization of thiolated (dT)25 single-stranded DNA (ssDNA) on gold. When electron attenuation effects are accurately accounted for in the XPS analysis, the relative coverage values obtained by the two methods are in excellent agreement, and the absolute coverage can be calculated on the basis of the XPS data. The evolution of chemically specific spectral signatures during immobilization indicates that at lower coverages much of the DNA lies flat on the surface, with a substantial fraction of the thymine bases chemisorbed. At higher immobilization densities, the (dT) 25 film consists of randomly coiled ssDNA molecules each anchored via the thiol group and at possibly one or two other bases. We use two examples to demonstrate how the quantitative analysis can be applied to practical problems: the effects of different buffer salts on the immobilization efficiency; the immobilization kinetics. Buffers with divalent salts dramatically increase the efficiency of immobilization and result in very high surface densities (>5 × 10 13 / cm 2 ), densities that may only be possible if the divalent counterions induce strong attractive intermolecular interactions. In contrast with previous reports of alkanethiol adsorption kinetics on gold, ssDNA immobilization in 1 M phosphate buffer does not occur with Langmuir kinetics, a result attributable to rearrangement within the film that follows the initial adsorption.

Base-Dependent Competitive Adsorption of Single-Stranded DNA on Gold

“…We characterize the adsorption ex-situ using Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS), two methods that together provide unambiguous spectral signatures of the different bases and allow absolute surface densities to be determined. 6 We produced ssDNA films by soaking clean Au substrates in 1 µM solutions of unmodified homo-oligonucleotides (1 M NaCl-TE buffer) for 20 h (see Supporting Information). Adsorption of each individual homo-oligonucleotide produces a specific FTIR signature ( Figure 1) and a different surface density as determined by XPS.…”

“…Thiolated (dT) 25 probes adsorb on Au as randomly coiled molecules each anchored via the thiol group. 6 Thus, one would expect this mixture to result in (dA) 25 ‚(dT) 25 duplexes adsorbed via the thiolated 5′ end of the (dT) 25 . However, the extraordinary affinity of oligo(dA) for Au again produces a film composed largely of adsorbed (dA) 25 (Figure 2, bottom).…”

Quantitative Characterization of DNA Films by X-ray Photoelectron Spectroscopy

“…Naval Research Laboratory,4555 Overlook Avenue SW,Washington,DC,20375 8. PERFORMING ORGANIZATION REPORT NUMBER…”

“…However, if the film is thick and disordered or if the ordering and the dynamic dipole moments do not change throughout a series of samples, FTIR can be used for quantitative coverage measurements. Because the peak at 1714 cm -1 in the FTIR data corresponds to free thymine rings, 8 in a comparison with XPS data the chemisorbed components (the two lower BE N 1s components in Figure 2) must be excluded from the coverage analysis. Figure 8 shows a comparison of the DNA coverage determined by FTIR (integrated peak area between 1615 and 1800 cm -1 , Figure 7) and from the main N 1s component 62 of the XPS spectra (400.5-401.0 eV BE in Figure 2).…”

Hydration Induced Stress on DNA Monolayers Grafted on Microcantilevers

“…17,18 The final strand density in self-assembled monolayers plays a critical role in nanomechanical sensing 9 and it is indeed far from being fully controllable. 19,[20][21][22][23] One singularly limiting difficulty has been to accomplish the anchoring of the ssDNA probes with a well-defined uniform density and a standing up conformation. 19,24 For such purpose, sophisticated nanografting methods need to be applied.…”