Spectroscopic quantification of covalently immobilized oligonucleotides

Abstract

Quantitative determination of surface coverage, film thickness and molecular orientation of DNA oligomers covalently attached to aminosilane self-assembled monolayers has been obtained using complementary infrared and photoelectron studies. Spectral variations between surface immobilized oligomers of the different nucleic acids are reported for the first time. Carbodiimide condensation was used for covalent attachment of phosphorylated oligonucleotides to silanized aluminum substrates. Fourier transform infrared (FTIR) spectroscopy and x-ray photoelectron spectroscopy (XPS) were used to characterize the surfaces after each modification step. Infrared reflection-absorption spectroscopy of covalently bound DNA provides orientational information. Surface density and layer thickness are extracted from XPS data. The surface density of immobilized DNA, 2-3 (×10 13 ) molecules cm −2 , was found to depend on base composition. Comparison of antisymmetric to symmetric phosphate stretching band intensities in reflection-absorption spectra of immobilized DNA and transmission FTIR spectra of DNA in KBr pellet indicates that the sugar-phosphate backbone is predominantly oriented with the sugar-phosphate backbone lying parallel to the surface, in agreement with the 10-20Å DNA film thickness derived from XPS intensities.

Thymine/adenine diblock-oligonucleotide monolayers and hybrid brushes on gold: a spectroscopic study

“…This seeming disagreement can be explained by the stronger attenuation of the photoelectron signal from the d(A) block serving as the molecular anchor and located, partly or almost completely, underneath the d(T) block. Note that the attenuation is especially strong at the kinetic energy of the N 1s photoelectrons (~180 eV; an attenuation length of 0.84 nm [33]), which explains why this effect was less pronounced in previous work relying on laboratory XPS systems [13,24,25] which have much higher excitation energy and, subsequently, higher photoelectron kinetic energies. Note also that the respective attenuation factor (~2.5) does not change significantly with the increasing length of the d(A) block, which contradicts the idea of the L-shape adsorption model and suggests that the exact adsorption geometry of the A n T 15 molecules depends on the molecular composition.…”

“…Techniques such as X-ray photoelectron spectroscopy (XPS) [13,14], near-edge X-ray absorption fine structure (NEXAFS) spectroscopy [15][16][17][18], time-of-flight secondary ion mass spectrometry (ToF-SIMS) [19][20][21], sum-frequency-generation (SFG) spectroscopy [22,23], and infrared reflection-absorption spectroscopy (IRRAS) [24,25] can be used to determine composition, content, molecular orientation and hybridization behavior of DNA assemblies. Yet despite the large amounts of potential information obtainable through use of these methods, the interpretation of spectra from biological samples, including surface-bound DNA, can be difficult, often resulting in the restriction of the use of these techniques to simplified or relatively straightforward systems.…”

Interaction of Self-Assembled Monolayers of DNA with Electrons: HREELS and XPS Studies

“…This discrepancy between the conclusions based on the two techniques might arise from the fact that each of the techniques is sensitive to a different region of the monolayer. Whereas HREELS probes mainly the end of the molecules facing the vacuum, as most processes are region position ss-poly(C) ss-poly(A) ss-poly(T) literature [29][30][31][32][33][34][35] due to impact mechanisms, XPS probes the coverage on the gold itself. It is quite possible that, although the layer is not dense and fully ordered near the substrate, the long molecules arrange themselves in such a way that their tails, facing the vacuum, are well-ordered.…”

Heterogeneous oligonucleotide-hybridization assay based on hot electron-induced electrochemiluminescence of a rhodamine label at oxide-coated aluminum and silicon electrodes

“…Aluminum electrodes covered with native, 2-3 nm thick oxide layer [33] and p-type silicon electrodes coated with 4 nm thick, thermally grown oxide layer were used in this study [13]. Fig.…”

“…Self-assembled aminosilane films have been deposited on a wide variety of hydroxylated surfaces, including glass [24][25][26], silicon dioxide [27][28][29], silicon nitride [30], indiumtin-oxide [31] and aluminum oxide [32,33]. The requirement for silane deposition is the presence of OH-groups on the surface, which allows formation of Si O bond between surface oxygen and silicon in the silanization reagent.…”