Angle-resolved XPS analysis and characterization of monolayer and multilayer silane films for DNA coupling to silica.

We measure silane density and Sulfo-EMCS cross-linker coupling efficiency on aminosilane films by high-resolution X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) measurements. We then characterize DNA immobilization and hybridization on these films by (32)P-radiometry. We find that the silane film structure controls the efficiency of the subsequent steps toward DNA hybridization. A self-limited silane monolayer produced from 3-aminopropyldimethylethoxysilane (APDMES) provides a silane surface density of ~3 nm(-2). Thin (1 h deposition) and thick (19 h deposition) multilayer films are generated from 3-aminopropyltriethoxysilane (APTES), resulting in surfaces with increased roughness compared to the APDMES monolayer. Increased silane surface density is estimated for the 19 h APTES film, due to a ∼32% increase in surface area compared to the APDMES monolayer. High cross-linker coupling efficiencies are measured for all three silane films. DNA immobilization densities are similar for the APDMES monolayer and 1 h APTES. However, the DNA immobilization density is double for the 19 h APTES, suggesting that increased surface area allows for a higher probe attachment. The APDMES monolayer has the lowest DNA target density and hybridization efficiency. This is attributed to the steric hindrance as the random packing limit is approached for DNA double helices (dsDNA, diameter ≥ 2 nm) on a plane. The heterogeneity and roughness of the APTES films reduce this steric hindrance and allow for tighter packing of DNA double helices, resulting in higher hybridization densities and efficiencies. The low steric hindrance of the thin, one to two layer APTES film provides the highest hybridization efficiency of nearly 88%, with 0.21 dsDNA/nm(2). The XPS data also reveal water on the cross-linker-treated surface that is implicated in device aging.

High-resolution X-ray photoelectron spectroscopy of mixed silane monolayers for DNA attachment.

The amine density of 3-aminopropyldimethylethoxysilane (APDMES) films on silica is controlled to determine its effect on DNA probe density and subsequent DNA hybridization. The amine density is tailored by controlling the surface reaction time of (1) APDMES, or (2) n-propyldimethylchlorosilane (PDMCS, which is not amine terminated) and then reacting it with APDMES to form a mixed monolayer. High-resolution X-ray photoelectron spectroscopy (XPS) is used to quantify silane surface coverage of both pure and mixed monolayers on silica; the XPS data demonstrate control of amine density in both pure APDMES and PDMCS/APDMES mixed monolayers. A linear correlation between the atomic concentration of N atoms from the amine and Si atoms from the APDMES in pure APDMES films allows us to calculate the PDMCS/APDMES ratio in the mixed monolayers. Fluorescence from attached DNA probes and from hybridized DNA decreases as the percentage of APDMES in the mixed monolayer is decreased by dilution with PDMCS.

Mutational analysis of the stator subunit E of the yeast V-ATPase.

Subunit E is a component of the peripheral stalk(s) that couples membrane and peripheral subunits of the V-ATPase complex. In order to elucidate the function of subunit E, site-directed mutations were performed at the amino terminus and carboxyl terminus. Except for S78A and D233A/T202A, which exhibited V(1)V(o) assembly defects, the function of subunit E was resistant to mutations. Most mutations complemented the growth phenotype of vma4Delta mutants, including T6A and D233A, which only had 25% of the wild-type ATPase activity. Residues Ser-78 and Thr-202 were essential for V(1)V(o) assembly and function. The mutation S78A destabilized subunit E and prevented assembly of V(1) subunits at the membranes. Mutant T202A membranes exhibited 2-fold increased V(max) and about 2-fold less of V(1)V(o) assembly; the mutation increased the specific activity of V(1)V(o) by enhancing the k(cat) of the enzyme 4-fold. Reduced levels of V(1)V(o) and V(o) complexes at T202A membranes suggest that the balance between V(1)V(o) and V(o) was not perturbed; instead, cells adjusted the amount of assembled V-ATPase complexes in order to compensate for the enhanced activity. These results indicated communication between subunit E and the catalytic sites at the A(3)B(3) hexamer and suggest potential regulatory roles for the carboxyl end of subunit E. At the carboxyl end, alanine substitution of Asp-233 significantly reduced ATP hydrolysis, although the truncation 229-233Delta and the point mutation K230A did not affect assembly and activity. The implication of these results for the topology and functions of subunit E within the V-ATPase complex are discussed.