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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.

We describe the use of self-assembled films of thiolated (dT)25 single-stranded DNA (ssDNA) on gold as a model system for quantitative characterization of DNA films by X-ray photoelectron spectroscopy (XPS). We evaluate the applicability of a uniform and homogeneous overlayer-substrate model for data analysis, examine model parameters used to describe DNA films (e.g., density and electron attenuation length), and validate the results. The model is used to obtain quantitative composition and coverage information as a function of immobilization time. We find that when the electron attenuation effects are properly included in the XPS data analysis, excellent agreement is obtained with Fourier transform infrared (FTIR) measurements for relative values of the DNA coverage, and the calculated absolute coverage is consistent with a previous radiolabeling study. Based on the effectiveness of the analysis procedure for model (dT)25 ssDNA films, it should be generally valid for direct quantitative comparison of DNA films prepared under widely varying conditions.

We describe self-assembly of ssDNA brushes that exploits the intrinsic affinity of adenine nucleotides (dA) for gold surfaces. The grafting density and conformation of these brushes is deterministically controlled by the length of the anchoring dA sequences, even in the presence of thymine nucleotides (dT) FTIR ͉ gold ͉ immobilization ͉ oligonucleotides ͉ x-ray photoelectron spectroscopy T he properties of surfaces functionalized with ssDNA exhibit a remarkable richness that underlies the versatility of these surfaces in a wide range of applications. The ssDNA brushes described in this work offer unique properties for two types of applications: control of nanoscale self-assembly (1, 2) and design and operation of biosensors (3-8). In general, a critical attribute of a ssDNA-modified surface is efficient and reproducible hybridization with target DNA. Model studies using thiolated DNA probes have shown that efficient hybridization occurs when the spacing between immobilized DNA probes is large and the orientation of the probes is upright (4-11). Unfortunately, reproducible preparation of DNA films possessing both of these qualities remains challenging (12-14). For example, it is generally observed that when the grafting density is low (Ͻ10 13 cm Ϫ2 ), i.e., the spacing between probes is comparable to their length, DNA immobilizes in a flat conformation through nonspecific adsorption (9,15,16). This observation can be largely explained by conventional polyelectrolyte brush theory, which predicts that negatively charged DNA strands should only assume upright conformations in densely packed films, where repulsive interactions force the strands to extend away from the surface (7)..Surface passivation is a common strategy used to decouple the DNA conformation from grafting density. Passivation prevents nonspecific interactions between the surface and DNA or other biomolecules, which enables widely spaced DNA probes to maintain an upright orientation. In a common implementation of this strategy, films of thiol-anchored DNA can be exposed to a solution containing a competing molecule, such as mercaptohexanol (MCH), which displaces most of the DNA from the surface and forces the remaining DNA strands into an upright conformation (10,(17)(18)(19). Alternatively, grafting densities can be adjusted by coupling functionalized DNA to a bifunctional self-assembled monolayer (6).Our objective in this study is to control independently and deterministically both the conformation and grafting density of DNA. We realize this objective by introducing anchoring sequences of adenine nucleotides (dA), which in our previous work have been shown to have a high intrinsic affinity for gold surfaces (20). Here, the function of the adenine blocks [(dA)] extends, however, beyond simple anchoring: They preferentially bind to gold surfaces and block nearly all of the adsorption sites, preventing nonspecific binding of other sequences to these surfaces (an effect similar to that of using the MCH posttreatment or a bifunctional self-assembled mon...

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We have studied the formation of self-assembled monolayers (SAMs) of n-alkanethiols on platinum thin films using X-ray photoelectron spectroscopy (XPS), reflection-absorption infrared spectroscopy (RAIRS), spectroscopic ellipsometry (SE), and contact angle (CA) measurements. Specifically, SAMs of 1-hexanethiol, 1-dodecanethiol, and 1-octadecanethiol were grown on polycrystalline Pt films, and the effects of Pt surface preparation, deposition conditions, and solvent treatments on the initial quality and stability of the monolayer in air were investigated. The SAMs prepared under ambient conditions on piranha-cleaned and UV/ozone-cleaned substrates were compared to monolayers formed on template-stripped Pt in an inert atmosphere. We found that alkanethiols deposited from 1 mM ethanolic solutions on piranha-cleaned Pt formed densely packed monolayers in which alkyl chains were oriented close to the surface normal. Stored in the laboratory ambient, these monolayers were unchanged over about 1 week but were largely oxidized in about 1 month. No evidence was found of molecules being weakly bound within the monolayer or having undergone C-S bond scission; however, three distinct sulfur states were observed for all samples in the XPS of the S 2p region. The lowest-and highest-binding-energy components are assigned to alkylthiolate and partially oxidized alkylthiolate species, respectively. The remaining S 2p component (approximately one-third of the sulfur layer), intermediate in binding energy between the other two components, is attributed to a chemisorbed species with a S binding configuration distinct from the majority alkylthiolate: for example, S bound to Pt bound to O, S with a different Pt coordination number, or S in an adsorbed disulfide.

Lysenin is a novel protein derived from coelomic fluid of the earthworm Eisenia foetida, which specifically recognizes sphingomyelin and induces cytolysis. The mechanism underlying lysenin-induced cell lysis has not been clarified. In this report we studied the interaction of lysenin with red blood cells as well as artificial liposomes. Our results showed that lysenin bound membranes and assembled to SDS-resistant oligomers in a sphingomyelin-dependent manner, leading to the formation of pores with a hydrodynamic diameter of ϳ3 nm. Antibody scanning analysis suggested that the Cterminal region of lysenin was exposed, whereas the N-terminal was hidden in the isolated oligomer complex. Differential scanning calorimetry revealed that lysenin interacted with both hydrophilic head group and hydrophobic hydrocarbon tails of sphingomyelin. Oligomerization but not binding was affected by the amide-linked fatty acid composition of sphingomyelin, suggesting the role of membrane fluidity in the oligomerization step.

The serum glycoprotein fetuin-A is an important inhibitor of extraosseous calcification. The importance of fetuin-A has been confirmed in fetuin-A null mice, which develop widespread extraosseous calcification including the kidney. However, the mechanism how fetuin-A protects kidneys from nephrocalcinosis remains uncertain. Here, we demonstrate that intratubular fetuin-A plays a role in the prevention of nephrocalcinosis in the proximal tubules. Although normal rat kidney did not express mRNA for fetuin-A, we found punctate immunohistochemical staining of fetuin-A mainly in the S1 segment of the proximal tubules. The staining pattern suggested that fetuin-A passed through the slit diaphragm, traveled in the proximal tubular lumen, and was introduced into proximal tubular cells by megalin-mediated endocytosis. To test this hypothesis, we inhibited the function of megalin by intravenous injection of histidine-tagged soluble receptor-associated protein (His-sRAP), a megalin inhibitor. His-sRAP injection diminished fetuin-A staining in the proximal tubules and led to urinary excretion of fetuin-A. We further analyzed the role of fetuin-A in nephrocalcinosis. Continuous injection of parathyroid hormone (PTH) 1–34 induced nephrocalcinosis mainly in the proximal tubules in rats. His-sRAP retained fetuin-A in renal tubular lumen and thereby protected the kidneys of PTH-treated rats from calcification. Our findings suggest that tubular luminal fetuin-A works as a natural inhibitor against calcification in the proximal tubules under PTH-loaded condition.