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Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HSssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s → π* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HSssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex milieu (i.e., serum) were characterized by surface plasmon resonance (SPR) and 32 P-radiometric assays and reported in a related study Methods for surface-immobilizing single-strand nucleic acids that preserve their original hybridization specificity with minimized nonspecific interactions remain an important goal for improving the performance of DNA microarray and biosensor applications. As summarized in a recent review, 1 nucleic acid hybridization behavior observed between complementary probe and target DNA molecules in bulk solution differ from identical hybridization at a solid-liquid * Corresponding author. interface. In surface hybridization, nonspecific probe-surface interactions, electrostatic forces, and steric issues between adjacent DNA probes influence DNA target hybridization efficiency and capacity. For example, nucleotide primary amines on nonhybridized DNA segments can interact (e.g., covalently 2 or by acid-base adsorption) with the surface, becoming unavailable to hybridize with target DNA molecules. Effects of immobilized DNA p...

The self-assembly of streptavidin onto biotinylated alkylthiolate monolayers on gold has served as an important model system for protein immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled monolayers (SAMs) containing biotinylated and diluent alkylthiolates and their use to specifically immobilize streptavidin. X-ray photoelectron spectroscopy (XPS), angle-resolved XPS (ARXPS), near-edge X-ray absorption fine structure (NEXAFS), and surface plasmon resonance (SPR) have been used to characterize the films produced on gold from a range of binary mixtures of a biotinylated alkylthiol (BAT) and either a C16 methyl-terminated thiol (mercaptohexadecane, MHD) or a C11-oligo(ethylene glycol)-terminated (OEG) thiol in ethanol. The correlation between the solution mole fraction of BAT and its surface mole fraction (χBAT,sur) indicates that it adsorbs ∼4-fold faster than OEG but slightly slower than MHD. ARXPS analysis demonstrates that the biotin terminus of the BAT is exposed at the surface of mixed monolayers with χBAT,sur < 0.5 but is randomly distributed through BAT-rich films. Thus, the OEG diluent not only adds nonfouling properties but induces an improved concentration of biotin at the surface and reduces the exposure of the methylene segments of BAT. NEXAFS characterization demonstrates that pure OEG and mixed BAT/OEG SAMs do not show significant anisotropy in C−C bond orientation, in contrast to MHD and mixed BAT/MHD SAMs, whose aliphatic segments exhibit pseudo-crystalline packing. SPR measurements of streptavidin binding to and competitive dissociation from the different mixed SAMs indicate that streptavidin binds both specifically and nonspecifically to the BAT/MHD SAMs but purely specifically to BAT/OEG SAMs with χBAT,sur < 0.5. For BAT/OEG mixtures with χBAT,sur = 0.1−0.5, specifically bound streptavidin coverages of ∼80% of the C(2,2,2) two-dimensional streptavidin crystalline density (∼280 ng/cm2) can be reproducibly achieved. These composite results clarify the relationship between the specificity of streptavidin recognition and the surface architecture and properties of the mixed SAMs.

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