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Poly(ethylene glycol) (PEG) nanofilms are used to avert the nonspecific binding of biomolecules on substrate surfaces in biomedicine and bioanalysis including modern fluorescence-based DNA sensing and sequencing chips. A fundamental and coherent understanding of the interactions between fluorophore-tagged DNA, PEG-films, and substrates in terms of molecular and energetic factors is, however, missing. Here we explore a large parameter space to elucidate how PEG layers passivate metal oxide surfaces against Cy3-labeled DNA probes. The driving force for probe adsorption is found to be the affinity of the fluorophore to the substrate, while the high-quality PEG films prevent adsorption to bare ITO surfaces. The amount of nonrepelled, surface-bound DNA strongly depends on oligonucleotide size, PEG chain length, and incubation temperature. To explain these observations, we develop an experimentally validated theory to provide a microscopic picture of the PEG layer and show that adsorbed DNA molecules reside within the film by end-tethering the fluorophore to the ITO surface. To compensate for the local accumulation of negatively charged DNA, counterions condense on the adsorbed probes within the layer. The model furthermore explains that surface passivation is governed by the interdependence of molecular size, conformation, charge, ion condensation, and environmental conditions. We finally report for the first time on the detailed thermodynamic values that show how adsorption results from a balance between large opposing energetic factors. The insight of our study can be applied to rationally engineer PEG nanolayers for improved functional performance in DNA analysis schemes and may be expanded to other polymeric thin films.

We present a generic and flexible method to nanopattern biomolecules on surfaces. Carbon-containing nanofeatures are written at variable diameter and spacing by a focused electron beam on a poly(ethylene glycol) (PEG)-coated glass substrate. Proteins physisorb to the nanofeatures with remarkably high contrast factors of more than 1000 compared to the surrounding PEG surfaces. The biological activity of model proteins can be retained as shown by decorating avidin spots with biotinylated DNA, thereby underscoring the universality of the nano-biofunctionalized platform for the binding of other biotinylated ligands. In addition, biomolecule densities can be tuned over several orders of magnitude within the same array, as demonstrated by painting a microscale image with nanoscale pixels. We expect that these unique advantages open up entirely new ways to design biophysical experiments, for instance, on cells that respond to the nanoscale densities of activating molecules., (© 2012 American Chemical Society)

The bottom-up approach of DNA nano-biotechnology can create biomaterials with defined properties relevant for a wide range of applications. This report describes nanoscale DNA tetrahedra that are beneficial to the field of biosensing and the targeted immobilization of biochemical receptors on substrate surfaces. The DNA nanostructures act as immobilization agents that are able to present individual molecules at a defined nanoscale distance to the solvent thereby improving biomolecular recognition of analytes. The tetrahedral display devices are self-assembled from four oligonucleotides. Three of the four tetrahedron vertices are equipped with disulfide groups to enable oriented binding to gold surfaces. The fourth vertex at the top of the bound tetrahedron presents the biomolecular receptor to the solvent. In assays testing the molecular accessibility via DNA hybridization and protein capturing, tetrahedron-tethered receptors outperformed conventional immobilization approaches with regard to specificity and amount of captured polypeptide by a factor of up to seven. The bottom-up strategy of creating DNA tetrahedrons is also compatible with the top-down route of nanopatterning of inorganic substrates, as demonstrated by the specific coating of micro- to nanoscale gold squares amid surrounding blank or poly(ethylene glycol)-passivated glass surfaces. DNA tetrahedra can create biofunctionalized surfaces of rationally designed properties that are of relevance in analytical chemistry, cell biology, and single-molecule biophysics., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Single-molecule characterization is essential for ascertaining the structural and functional properties of bottom-up DNA nanostructures. Here we enlist three atomic force microscopy (AFM) techniques to examine tetrahedron-shaped DNA nanostructures that are functionally enhanced with small chemical tags. In line with their application for biomolecule immobilization in biosensing and biophysics, the tetrahedra feature three disulfide-modified vertices to achieve directed attachment to gold surfaces. The remaining corner carries a single bioligand that can capture and present individual cargo biomolecules at defined lateral nanoscale spacing. High-resolution AFM topographic imaging confirmed the directional surface attachment as well as the highly effective binding of individual receptor molecules to the exposed bioligands. Insight into the binding behavior at the single-molecule level was gained using molecular recognition force spectroscopy using an AFM cantilever tip with a tethered molecular receptor. Finally, simultaneous topographic and recognition imaging demonstrated the specific receptor-ligand interactions on individual tetrahedra. In summary, AFM characterization verified that the rationally designed DNA nanostructures feature characteristics to serve as valuable immobilization agents in biosensing, biophysics, and cell biology., (© 2011 American Chemical Society)

Native-protein nanolithography (NPNL) was used to fabricate stable bioactive arrays of viral receptor spots. The arrays were specific for the cognate virus and devoid of nonspecific protein and virus adsorption under physiologic conditions. The spot size ranged from 200 nm x 200 nm to 2 microm x 2 microm and up to 3 x 3 spots were arranged per array. With proper force adjustment in the patterning experiments, His(6)-tagged bovine serum albumin (BSA) molecules were selectively removed from the underlying self-assembled monolayer (SAM) while leaving the latter intact. Injection of His(6)-tagged very low density lipoprotein receptor (VLDLR-His(6)) constructs resulted in specific, oriented binding to the Ni(2+)-loaded bis-(nitrolotriacetic acid) (bis-NTA) groups to the re-exposed SAM areas. The arrays of viral receptors were used for the detection of human rhinovirus particles (serotype 2; HRV2) under native conditions by topographical imaging at high signal-to-noise ratio. The kinetic on-rate of the HRV2-VLDLR interaction was derived from the time-dependent binding of the virions to the VLDL receptor spots. No significant binding was observed for the major group virus HRV14 that uses the unrelated receptor ICAM-1.

We describe the formation and characterization of surface-passivating poly(ethylene glycol) (PEG) films on indium tin oxide (ITO) glass substrates. PEG chains with a molecular weight of 2000 and 5000 D were covalently attached to the substrates in a systematic approach using different coupling schemes. The coupling strategies included the direct grafting with PEG-silane, PEG-methacrylate, and PEG-bis(amine), as well as the two-step functionalization with aldehyde-bearing silane films and subsequent coupling with PEG-bis(amine). Elemental analysis by X-ray photoelectron spectroscopy (XPS) confirmed the successful surface modification, and XPS and ellipsometry provided values for film thicknesses. XPS and ellipsometry thickness values were almost identical for PEG-silane films but differed by up to 400% for the other PEG layers, suggesting a homogeneous layer for PEG-silane but an inhomogeneous distribution for other PEG coatings on the molecularly rough ITO substrates. Atomic force microscopy (AFM) and water contact angle goniometry confirmed the different degrees of surface homogeneity of the polymer films, with PEG-silane reducing the AFM rms surface roughness by 50% and the water contact angle hysteresis by 75% compared to uncoated ITO. The ability of the PEG layers to passivate the substrate against the nonspecific adsorption of biopolymers was tested using fluorescence-labeled immunoglobulin G and DNA oligonucleotides in combination with fluorescence microscopy. The results indicate a positive relationship between film density and homogeneity on one hand and the ability to passivate against biopolymer adhesion on the other hand. The most homogeneous layers prepared with PEG-silane reduced the nonspecific adsorption of fluorescence-labeled DNA by a factor of 300 compared to uncoated ITO. In addition, the study finds that the ratio of film thicknesses derived by ellipsometry and XPS is a useful parameter to quantify the structural integrity of PEG layers on molecularly rough ITO surfaces. The findings may be applied to characterize PEG or other polymeric films on similarly coarse substrates.

Indium tin oxide (ITO) substrates were modified with a layer of poly(amidoamine) (PAMAM) dendrimers to change their surface properties and, in particular, the substrates' work function. The functionalization procedure involved the electrostatic adsorption of positively charged PAMAM dendrimers of generation five onto negatively polarized ITO surfaces. Three different PAMAM dendrimers were used: PAMAM-NH2 and PAMAM-OH with terminal amine and hydroxyl groups, respectively, as well as Q-PAMAM-NH2, which had been prepared from PAMAM-NH2 by quaternization of the dendrimer's terminal and internal amine groups with methyl iodide. The resulting organic films were analyzed by contact angle goniometry, X-ray photoelectron spectroscopy, ellipsometry, and Kelvin probe force microscopy to confirm the presence of a dense layer. A Langmuir isotherm was derived from surface densities of fluorescence-labeled PAMAM-NH2 dendrimers from which we deduced an equilibrium binding constant, K(eq), of (1.3 +/- 0.3) x 10(5) M(-1). Kelvin probe measurements of the contact potential difference revealed a high reduction of the work function from 4.9 eV for bare ITO to 4.3 eV for ITO with a dense film of PAMAM-NH2 of generation five. PAMAM-OH and Q-PAMAM-NH2 resulted in slightly smaller work function changes. This study illustrates that the work function of ITO can be tuned by adlayers composed of PAMAM dendrimers.

Aldehyde functions are widely used for immobilization of biomolecules on glass surfaces but have found little attention for biofunctionalization of self-assembled monolayers (SAMs) on gold, due to interference between thiol and aldehyde functions. This problem was recently solved by synthesis of an alkanethiol that carried a vicinal diol group [Jang et al. (2003) Nano Lett. 3, 691-694]. The latter served as a latent aldehyde function that was unmasked by short exposure of the vicinal diol-terminated SAM to aqueous periodate. However, the synthesis of the new vicinal diol-terminated alkane thiol was time-consuming and had an overall yield of approximately 3.5%. In the present study, a general modular strategy was introduced by which SAM components with vicinal diol functions were rapidly synthesized with high yield: this was accomplished by amide bond formation between a SAM-forming carboxylic acid (exemplified by lipoic acid and 16-mercaptohexadecanoic acid) with 3-aminopropane-1,2-diol, using suitable protecting groups. The disulfide or free thiol group afforded SAM formation on gold and, after periodate oxidation of the vicinal diol functions, proteins were covalently bound via their lysine residues. At 1 mg/mL protein concentration, complete surface coverage was reached within minutes. No further protein was bound by nonspecific adsorption, but cognate proteins were specifically bound with high capacity. Pyrogallol-O-hexadecanoic acid and 10-undecenoic acid were also coupled with 3-aminopropane-1,2-diol by amide bond formation, thereby producing latent aldehyde-containing SAM components for metal oxides and hydrogen-terminated silicon, respectively, to show the general usefulness of the new synthetic design.

We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration--corresponding to only 39,000 molecules in the sample solution--and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10(4) cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.

Surfaces carrying a dense layer of poly(ethylene glycol) (PEG) were prepared, characterized, and tested as substrates for DNA oligonucleotide microarrays. PEG bis(amine) with a molecular weight of 2000 was grafted onto silanized glass slides bearing aldehyde groups. After grafting, the terminal amino groups of the PEG layer were derivatized with the heterobifunctional cross-linker succinimidyl 4-[p-maleimidophenyl]butyrate to permit the immobilization of thiol-modified DNA oligonucleotides. The stepwise chemical modification was validated with X-ray photoelectron spectroscopy. Goniometry indicated that the PEG grafting procedure reduced surface inhomogeneities present after the silanization step, while atomic force microscopy and ellipsometry confirmed that the PEG layer was dense and monomolecular. Hybridization assays using DNA oligonucleotides and fluorescence imaging showed that PEG grafting improved the yield in hybridization 4-fold compared to non-PEGylated maleimide-derivatized surfaces. In addition, the PEG layer reduced the nonspecific adsorption of DNA by a factor of up to 13, demonstrating that surfaces with a dense PEG layer represent suitable substrates for DNA oligonucleotide microarrays.