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This study assesses the capability of high-resolution surface analytical tools to distinguish immobilized antibody orientations on patterned surfaces designed for antibody affinity capture. High-fidelity, side-by-side copatterning of protein A (antibody Fc domain affinity reagent) and fluorescein (antibody Fab domain hapten) was achieved photolithographically on commercial amine-reactive hydrogel polymer surfaces. This was verified from fuorescence imaging using fluorescently labeled protein A and intrinsic fluorescence from fluorescein. Subsequently, dye-labeled murine antifluorescein antibody (4-4-20) and antibody Fab and Fc fragments were immobilized from solution onto respective protein A- and fluorescein- copatterned or control surfaces using antibody--ligand affinity interactions. Fluorescence assays support specific immobilization to fluorescein hapten- and protein A-patterned regions through antigen--antibody recognition and natural protein A--Fc domain interactions, respectively. Affinity-based antibody immobilization on the two different copatterned surfaces generated side-by-side full antibody 'heads-up' and 'tails-up' oriented surface patterns. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis, sensitive to chemical information from the top 2 to 3 nm of the surface, provided ion-specific images of these antibody patterned regions, imaging and distinguishing characteristic ions from amino acids enriched in Fab domains for antibodies oriented in 'heads-up' regions, and ions from amino acids enriched in Fc domains for antibodies oriented in 'tails-up' regions. Principal component analysis (PCA) improved the distinct TOF-SIMS amino acid compositional and ion-specific surface mapping sensitivity for each 'heads-up' versus 'tails-up' patterned region. Characteristic Fab and Fc fragment immobilized patterns served as controls. This provides first demonstration of pattern-specific, antibody orientation-dependent surface maps based on antibody domain- and structure-specific compositional differences by TOF-SIMS analysis. Since antibody immobilization and orientation are critical to many technologies, orientation characterization using TOF-SIMS could be very useful and convenient for immobilization quality control and understanding methods for improving the performance of antibody-based surface capture assays. 10.1021/ac902964q

X-ray photoelectron (XPS) is used as a surface-attached hematic complex on the native oxide of silicon for the direct detection of NO. The silicon platform has enabled the implementation of the surface-modification strategy on a silicon-based device for direct electrical detection of NO.

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAPS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy. [ABSTRACT FROM AUTHOR]

The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that anionic lipid membranes can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40s presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40s strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

This study assesses the capability of high-resolution surface analytical tools to distinguish immobilized antibody orientations on patterned surfaces designed for antibody affinity capture. High-fidelity, side-by-side copatterning of protein A (antibody Fc domain affinity reagent) and fluorescein (antibody Fab domain hapten) was achieved photolithographically on commercial aminereactive hydrogel polymer surfaces. This was verified from fluorescence imaging using fluorescently labeled protein A and intrinsic fluorescence from fluorescein. Subsequently, dye-labeled murine antifluorescein antibody (4-4-20) and antibody Fab and Fc fragments were immobilized from solution onto respective protein Aand fluoresceincopatterned or control surfaces using antibody-ligand affinity interactions. Fluorescence assays support specific immobilization to fluorescein haptenand protein A-patterned regions through antigen-antibody recognition and natural protein A-Fc domain interactions, respectively. Affinity-based antibody immobilization on the two different copattemed surfaces generated sideby-side full antibody `heads-up" and `tails-up" oriented surface patterns. Time-of-ifight secondary ion mass spectrometry (TOF-SIMS) analysis, sensitive to chemical information from the top 2 to 3 nm of the surface, provided ion-specific images of these antibody patterned regions, imaging and distinguishing characteristic ions from amino acids enriched in Fab domains for antibodies oriented in `heads-up" regions, and ions from amino acids enriched in Fe domains for antibodies oriented in "tails-up" regions. Principal component analysis (PCA) improved the distinct TOF-SIMS amino acid compositional and ionspecific surface mapping sensitivity for each "heads-up" versus "tails-up" patterned region. Characteristic Fab and Fe fragment immobilized patterns served as controls. This provides first demonstration of pattern-specific, antibody orientation-dependent surface maps based on antibody domainand structure-specific compositional differences byTOF-SIMS analysis. Since antibody immobilization and orientation are critical to many technologies, orientation characterization using TOF-SIMS could be vety useful and convenient for immobilization quality control and understanding methods for improving the performance of antibody-based surface capture assays. [ABSTRACT FROM AUTHOR]

Pharmaceutical powder engineering often involves forming interfaces between the drug and a suitable polymer. The structure at the interface plays a critical role in the properties and performance of the composite. However, interface structures have not been well understood due to a lack of suitable characterization tool. In this work, we have used ellipsometry and neutron reflectometry to characterize the structure of such interfaces in detail. Ellipsometry provided a quick estimate of the number of layers and their thicknesses, whereas neutron reflectometry provided richer structural information such as density, thickness, roughness, and intermixing of different layers. The combined information allowed us to develop an accurate model about the layered structure and provided information about intermixing of different layer components. Systematic use of these characterization techniques on several model systems suggests that the nature of the polymer had a small effect on the interfacial structure, while the solvent used in polymer coating had a large effect. These results provide useful information on the efforts of engineering particle properties through the control of the interfacial chemistry.

A study of the interaction of four endoglucanases with amorphous cellulose films by neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) is reported. The endoglucanases include a mesophilic fungal endoglucanase (Cel45A from H. insolens), a processive endoglucanase from a marine bacterium (Cel5H from S. degradans ), and two from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima ). The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. The endoglucanases displayed highly diverse behavior. Cel45A and Cel5H, which possess carbohydrate-binding modules (CBMs), penetrated and digested within the bulk of the films to a far greater extent than Cel9A and Cel5A, which lack CBMs. While both Cel45A and Cel5H were active within the bulk of the films, striking differences were observed. With Cel45A, substantial film expansion and interfacial broadening were observed, whereas for Cel5H the film thickness decreased with little interfacial broadening. These results are consistent with Cel45A digesting within the interior of cellulose chains as a classic endoglucanase, and Cel5H digesting predominantly at chain ends consistent with its designation as a processive endoglucanase.

Silicon photonic microring resonators have established their potential for label-free and low-cost biosensing applications. However, the long-term performance of this optical sensing platform requires robust surface modification and biofunctionalization. Herein, we demonstrate a conjugation strategy based on an organophosphonate surface coating and vinyl sulfone linker to biofunctionalize silicon resonators for biomolecular sensing. To validate this method, a series of glycans, including carbohydrates and glycoconjugates, were immobilized on divinyl sulfone (DVS)/organophosphonate-modified microrings and used to characterize carbohydrate-protein and norovirus particle interactions. This biofunctional platform was able to orthogonally detect multiple specific carbohydrate-protein interactions simultaneously. Additionally, the platform was capable of reproducible binding after multiple regenerations by high-salt, high-pH, or low-pH solutions and after 1 month storage in ambient conditions. This remarkable stability and durability of the organophosphonate immobilization strategy will facilitate the application of silicon microring resonators in various sensing conditions, prolong their lifetime, and minimize the cost for storage and delivery; these characteristics are requisite for developing biosensors for point-of-care and distributed diagnostics and other biomedical applications. In addition, the platform demonstrated its ability to characterize carbohydrate-mediated host-virus interactions, providing a facile method for discovering new antiviral agents to prevent infectious disease.

Improving the efficiency of enzymatic hydrolysis of cellulose is one of the key technological hurdles to reduce the cost of producing ethanol and other transportation fuels from lignocellulosic material. A better understanding of how soluble enzymes interact with insoluble cellulose will aid in the design of more efficient enzyme systems. We report a study involving neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) of the interaction of a fungal enzyme extract ( T. viride ) and an endoglucanse from A. niger with amorphous cellulose films. The use of amorphous cellulose is motivated by that the fact that several biomass pretreatments currently under investigation disrupt the native crystalline structure of cellulose and increase the amorphous content. NR reveals the profile of water through the film at nanometer resolution and is highly sensitive to interfacial roughness, whereas QCM-D provides changes in mass and film stiffness. NR can be performed using either H(2)O- or D(2)O-based aqueous reservoirs. NR measurement of swelling of a cellulose film in D(2)O and in H(2)O revealed that D/H exchange on the cellulose chains must be taken into account when a D(2)O-based reservoir is used. The results also show that cellulose films swell slightly more in D(2)O than in H(2)O. Regarding enzymatic digestion, at 20 °C in H(2)O buffer the T. viride cocktail rapidly digested the entire film, initially roughening the surface, followed by penetration and activity throughout the bulk of the film. In contrast, over the same time period, the endoglucanase was active mainly at the surface of the film and did not increase the surface roughness.

Organophosphonic acid self-assembled monolayers (SAMs) on oxide surfaces have recently seen increased use in electrical and biological sensor applications. The reliability and reproducibility of these sensors require good molecular organization in these SAMs. In this regard, packing, order, and alignment in the SAMs is important, as it influences the electron transport measurements. In this study, we examine the order of hydroxyl- and methyl-terminated phosphonate films deposited onto silicon oxide surfaces by the tethering by aggregation and growth method using complementary, state-of-art surface characterization tools. Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy and in situ sum frequency generation (SFG) spectroscopy are used to study the order of the phosphonate SAMs in vacuum and under aqueous conditions, respectively. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry results show that these samples form chemically intact monolayer phosphonate films. NEXAFS and SFG spectroscopy showed that molecular order exists in the octadecylphosphonic acid and 11-hydroxyundecylphosphonic acid SAMs. The chain tilt angles in these SAMs were approximately 37° and 45°, respectively.

We investigated hydroxyalkylphosphonate monolayers as a novel platform for the biofunctionalization of silicon-based field effect sensor devices. This included a detailed study of the thin film properties of organophosphonate films on Si substrates using several surface analysis techniques, including AFM, ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), X-ray reflectivity, and current-voltage characteristics in electrolyte solution. Our results indicate the formation of a dense monolayer on the native silicon oxide that has excellent passivation properties. The monolayer was biofunctionalized with 12 mer peptide nucleic acid (PNA) receptor molecules in a two-step procedure using the heterobifunctional linker, 3-maleimidopropionic-acid-N-hydroxysuccinimidester. Successful surface modification with the probe PNA was verified by XPS and contact angle measurements, and hybridization with DNA was determined by fluorescence measurements. Finally, the PNA functionalization protocol was translated to 2 microm long, 100 nm wide Si nanowire field effect devices, which were successfully used for label-free DNA/PNA hybridization detection.

The attachment of a bifunctional iodo-organo-phosphinate compound to gold (Au) surfaces via chemisorption of the iodine atom is described and used to chelate a redox-active metal cluster via the phosphinate group. XPS, AFM, and electrochemical measurements show that (4-iodo-phenyl)phenyl phosphinic acid (IPPA) forms a tightly bound self-assembled monolayer (SAM) on Au surfaces. The surface coverage of an IPPA monolayer on Au was quantified by an electrochemical method and found to be 0.40 +/- 0.03 nmol/cm2, roughly corresponding to 0.4 monolayers. We show that the Au/IPPA SAM, but not the underivatized Au, adsorbs Mn4O4(Ph2PO2)6 from solution by a phosphinate exchange reaction to yield Au/IPPA/Mn4O4(Ph2PO2)5 SAM. The resulting SAM is firmly bound and not removed by sonication, as confirmed by manganese XPS (Mn 2p1/2) and by AFM. Electrochemistry confirms that Mn4O4(Ph2PO2)6 is anchored on the Au/IPPA surface and that redox chemistry can be mediated between the electrode and the surface-attached complex. Mn4O4(Ph2PO2)6 contains the reactive Mn4O46+ cubane core, a redox-active bioinspired catalyst.

Phosphonate-steel interactions have been industrially significant for decades, but details of the phosphonate-steel interface have not yet been characterized. Self-assembled monolayers of phosphonic acids were formed on stainless steel 316L by room-temperature solution deposition. The acids are covalently bound to the surface as phosphonates in a bidentate manner, as determined by diffuse reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Complete coverage of the surface is confirmed by contact angle measurement and atomic force microscopic imaging. This method of monolayer formation contrasts the requirement for heating and long reaction times found to be necessary to form phosphonate monolayers on other metal oxide substrates, such as titanium and silicon.

Differential charging is often regarded as a problem in X-ray photoelectron spectroscopic studies, especially for insulating or partially conducting samples. Application of a positive bias can reduce the effect of differential charging by attracting stray electrons from the system, thereby compensating for the electron loss. On the other hand, differential charging effect can be enhanced by the application of a negative bias to the sample during spectrum acquisition. The successful use of the differential charging technique to distinguish between multi- and monolayer organophosphonate films on oxide-covered silicon has been reported. A detailed description of this technique is now presented which shows how differential charging can be used as an important tool for the characterization of self-assembled films deposited on various surfaces. The dependence of this technique on the conductivity of the substrate has been investigated by studying the spectral behavior of the deposited films of phosphonic acid on conducting, semiconducting, and insulating samples (stainless steel, silicon, and glass). Application of either positive or negative dc electrical bias affects the carbon core-level (C1s) line shape and intensity, which is dependent on the atom's physical location above the surface.