Lara J. Gamble

The interaction of H2O with a TiO2(110) surface

Abstract The interaction of H2O with rutile TiO2(110) surfaces with different defect densities (oxygen vacancies) was studied with TPD, work function measurements and XPS. On the nearly perfect surface, a thermal desorption peak is observed at 250–300 K which is attributed to molecularly adsorbed H2O at Ti4+ sites based on its O(1s) peak position and work function change. The heat of adsorption of water in this state is estimated to be 71-9θ kJ/mol. The coverage of water in this state is estimated from O(1s) signals to be about one per unit cell, or one for every Ti4+ site. A tail of this peak which extends to 375 K is attributed to disproportionation of surface hydroxyl groups present in lower concentrations. Higher coverages of water give rise to a TPD peak at 170 K, which we attribute to water bound to bridging oxygen anion sites. Finally, multilayer water is populated, which desorbs in a peak at 160 K. Surface hydroxyls bound to thermally-induced oxygen vacancies of ∼ 1% concentration disproportionate to give a water TPD peak at ∼ 500 K.

Decomposition and protonation of surface ethoxys on TiO2 (110)

The decomposition and protonation of surface ethoxy groups on TiO2(110) has been studied as a function of surface hydroxyl or water coverage by TPD and XPS. Surface ethoxys were produced by dissociative adsorption of deuterated ethanol (EtOD), as well as tetraethoxysilane (TEOS). The effects of both preadsorption and post-adsorption of water (and hydroxyls) on the thermal reactions of these ethoxys are studied. Two different types of adsorbed ethoxy species are identified: (1) ethoxys which can readily be removed by combination with surface hydroxyl groups to desorb as ethanol gas from ∼250 to 400 K, and (2) ethoxys which cannot react with surface water or hydroxyls below ∼450 K in TPD, even when recooled, dosed with hydroxyls again and reheated. The first type is attributed to ethoxy groups bound to surface Ti atoms (TiOEt), protonated with a proton from a hydroxyl group formed by a “bridging oxygen” atom of the surface lattice, Obr. The second type is attributed to ethoxy groups bound at “bridging oxygen” vacancies in the surface lattice, Obr-Et. Such vacancies are formed when water is produced from two Obr-H species. The Obr-Et groups are removed at ∼ 650 K by decomposition, giving ethylene and ethanol in a 1:1 ratio. Product yields and a temperature shift found when using CD3CH2OH prove that this decomposition is initiated by β-hydride elimination. Ethylene evolution is, however, not concerted with β-hydrogen elimination. The TiOEt species also follows this path if insufficient hydroxyls are present to allow its full removal below 400 K. Migration of ethoxy and hydroxyl species between Ti4+ sites and surface oxygen vacancies is postulated to explain quantitative yields. Experiments on sputtered TiO2 support this model.

Probing the orientation and conformation of alpha-helix and beta-strand model peptides on self-assembled monolayers using sum frequency generation and NEXAFS spectroscopy.

The structure and orientation of amphiphilic alpha-helix and beta-strand model peptide films on self-assembled monolayers (SAMs) have been studied with sum frequency generation (SFG) vibrational spectroscopy and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. The alpha-helix peptide is a 14-mer, and the beta-strand is a 15-mer of hydrophilic lysine and hydrophobic leucine residues with hydrophobic periodicities of 3.5 and 2, respectively. These periodicities result in the leucine side chains located on one side of the peptides and the lysine side chains on the other side. The SAMs were prepared from the assembly of either carboxylic acid- or methyl-terminated alkyl thiols onto gold surfaces. For SFG studies, the deuterated analog of the methyl SAM was used. SFG vibrational spectra in the C-H region of air-dried peptides films on both SAMs exhibit strong peaks near 2965, 2940, and 2875 cm(-1) related to ordered leucine side chains. The orientation of the leucine side chains was determined from the phase of these features relative to the nonresonant gold background. The relative phase for both the alpha-helix and beta-strand peptides showed that the leucine side chains were oriented away from the carboxylic acid SAM surface and oriented toward the methyl SAM surface. Amide I peaks observed near 1656 cm(-1) for the alpha-helix peptide confirm that the secondary structure is preserved on both SAMs. Strong linear dichroism related to the amide pi* orbital at 400.8 eV was observed in the nitrogen K-edge NEXAFS spectra for the adsorbed beta-strand peptides, suggesting that the peptide backbones are oriented parallel to the SAM surface with the side chains pointing toward or away from the interface. For the alpha-helix the dichroism of the amide pi* is significantly weaker, probably because of the broad distribution of amide bond orientations in the alpha-helix secondary structure.

Probing the Orientation of Surface-Immobilized Protein G B1 Using ToF-SIMS Sum Frequency Generation and NEXAFS Spectroscopy

The ability to orient active proteins on surfaces is a critical aspect of many medical technologies. An important related challenge is characterizing protein orientation in these surface films. This study uses a combination of time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum frequency generation (SFG) vibrational spectroscopy, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to characterize the orientation of surface-immobilized Protein G B1, a rigid 6 kDa domain that binds the Fc fragment of IgG. Two Protein G B1 variants with a single cysteine introduced at either end were immobilized via the cysteine thiol onto maleimide-oligo(ethylene glycol)-functionalized gold and bare gold substrates. X-ray photoelectron spectroscopy was used to measure the amount of immobilized protein, and ToF-SIMS was used to measure the amino acid composition of the exposed surface of the protein films and to confirm covalent attachment of protein thiol to the substrate maleimide groups. SFG and NEXAFS were used to characterize the ordering and orientation of peptide or side chain bonds. On both substrates and for both cysteine positions, ToF-SIMS data showed enrichment of mass peaks from amino acids located at the end of the protein opposite to the cysteine surface position as compared with nonspecifically immobilized protein, indicating end-on protein orientations. Orientation on the maleimide substrate was enhanced by increasing pH (7.0-9.5) and salt concentration (0-1.5 M NaCl). SFG spectral peaks characteristic of ordered α-helix and β-sheet elements were observed for both variants but not for cysteine-free wild type protein on the maleimide surface. The phase of the α-helix and β-sheet peaks indicated a predominantly upright orientation for both variants, consistent with an end-on protein binding configuration. Polarization dependence of the NEXAFS signal from the N 1s to π* transition of β-sheet peptide bonds also indicated protein ordering, with an estimated tilt angle of inner β-strands of 40-50° for both variants (one variant more tilted than the other), consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces.

XPS, TOF-SIMS, NEXAFS, and SPR characterization of nitrilotriacetic acid-terminated self-assembled monolayers for controllable immobilization of proteins.

For immobilization of proteins onto surfaces in a specific and controlled manner, it is important to start with a well-defined surface that contains specific binding sites surrounded by a nonfouling background. For immobilizing histidine-tagged (his-tagged) proteins, surfaces containing nitrilotriacetic acid (NTA) headgroups and oligo(ethylene glycol) (OEG) moieties are a widely used model system. The surface composition, structure, and reactivity of mixed NTA/OEG self-assembled monolayers (SAMs) on Au substrates were characterized in detail using X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), time-of-flight secondary ion mass spectrometry (TOF-SIMS), and surface plasmon resonance (SPR) biosensoring. XPS results for sequential adsorption of NTA thiols followed by OEG thiols showed that OEG molecules were incorporated into an incompletely formed NTA monolayer until a complete mixed SAM was formed. The surface concentration of NTA headgroups was estimated to be 0.9-1.3 molecule/nm2 in the mixed NTA/OEG monolayers, compared to 1.9 molecule/nm2 in pure NTA monolayers. Angle-dependent XPS indicated NTA headgroups were slightly reoriented toward an upright position after OEG incorporation, and polarization-dependent NEXAFS results indicated increased ordering of the alkane chains of the molecules. Nitrogen-containing and OEG-related secondary ion fragments from the TOF-SIMS experiments confirmed the presence of NTA headgroups and OEG moieties in the monolayers. A multivariate peak intensity ratio was developed for estimating the relative NTA concentration in the outermost (10 A) of the monolayers. SPR measurements of a his-tagged, humanized anti-lysozyme variable fragment (HuLys Fv) immobilized onto Ni(II)-treated mixed NTA/OEG and pure NTA monolayers demonstrated the reversible, site-specific immobilization of his-tagged HuLys Fv (108-205 ng/cm2) with dissociation rates (koff) between 1.0 x 10-4 and 2.1 x 10-5 s-1, both depending on the NTA surface concentration and orientation. The monolayers without Ni(II) treatment exhibited low nonspecific adsorption of his-tagged HuLys Fv (<2 ng/cm2).

Simple surface modification of a titanium alloy with silanated zwitterionic phosphorylcholine or sulfobetaine modifiers to reduce thrombogenicity

Thrombosis and thromboembolism remain problematic for a large number of blood contacting medical devices and limit broader application of some technologies due to this surface bioincompatibility. In this study we focused on the covalent attachment of zwitterionic phosphorylcholine (PC) or sulfobetaine (SB) moieties onto a TiAl(6)V(4) surface with a single step modification method to obtain a stable blood compatible interface. Silanated PC or SB modifiers (PCSi or SBSi) which contain an alkoxy silane group and either PC or SB groups were prepared respectively from trimethoxysilane and 2-methacryloyloxyethyl phosphorylcholine (MPC) or N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine (SMDAB) monomers by a hydrosilylation reaction. A cleaned and oxidized TiAl(6)V(4) surface was then modified with the PCSi or SBSi modifiers by a simple surface silanization reaction. The surface was assessed with X-ray photoelectron spectroscopy (XPS), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and contact angle goniometry. Platelet deposition and bulk phase activation were evaluated following contact with anticoagulated ovine blood. XPS results verified successful modification of the PCSi or SBSi modifiers onto TiAl(6)V(4) based on increases in surface phosphorous or sulfur respectively. Surface contact angles in water decreased with the addition of hydrophilic PC or SB moieties. Both the PCSi and SBSi modified TiAl(6)V(4) surfaces showed decreased platelet deposition and bulk phase platelet activation compared to unmodified TiAl(6)V(4) and control surfaces. This single step modification with PCSi or SBSi modifiers offers promise for improving the surface hemocompatibility of TiAl(6)V(4) and is attractive for its ease of application to geometrically complex metallic blood contacting devices.

Modulus-dependent macrophage adhesion and behavior

Macrophage attachment and activation to implanted materials is crucial in determining the extent of acute and chronic inflammation, and biomaterials degradation. In an effort to improve implant performance, considerable attention has centered on altering material surface chemistry to modulate macrophage behavior. In this work, the influence of the modulus of a material on the behavior of model macrophages (i.e., human promonocytic THP-1 cells) was investigated. We synthesized interpenetrating polymer network (IPN) coatings with varying moduli to test the hypothesis that lower moduli surfaces attenuate THP-1 cell attachment and activation. The surface chemistry and moduli of the IPN coatings were characterized using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), respectively. THP-1 cells preferentially attached to stiffer coatings of identical surface chemistry, confirming that fewer macrophages attach to lower moduli surfaces. The secretion of human TNF-α, IL-10, IL-8 and IL-1β from THP-1 cells attached to the IPNs was measured to assess the concentration of both pro- and anti-inflammatory cytokines. The global amount of TNF-α released did not vary for IPN surfaces of different moduli; however, the amount of the pro-inflammatory cytokine IL-8 released demonstrated a biphasic response, where lower (approx. 1.4 kPa) and very high (approx. 348 kPa) moduli IPN surfaces attenuated IL-8 secretion. The different trends for TNF-α and IL-8 secretion highlight the complexity of the wound healing response, suggesting that there may not be a unique surface chemistry and substratum modulus combination that minimizes the pro-inflammatory cytokines produced by activated macrophages.

Structure and Order of Phosphonic Acid-Based Self-Assembled Monolayers on Si(100)

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.

Bioactive polymer grafting onto titanium alloy surfaces

Bioactive polymers bearing sulfonate (styrene sodium sulfonate, NaSS) and carboxylate (methylacrylic acid, MA) groups were grafted onto Ti6Al4V alloy surfaces by a two-step procedure. The Ti alloy surfaces were first chemically oxidized in a piranha solution and then directly subjected to radical polymerization at 70 degrees C in the absence of oxygen. The grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the toluidine blue colorimetric method. Toluidine blue results showed 1-5microgcm(-2) of polymer was grafted onto the oxidized Ti surfaces. Grafting resulted in a decrease in the XPS Ti and O signals from the underlying Ti substrate and a corresponding increase in the XPS C and S signals from the polymer layer. The ToF-SIMS intensities of the S(-) and SO(-) ions correlated linearly with the XPS atomic percent S concentrations and the ToF-SIMS intensity of the TiO(3)H(2)(-) ion correlated linearly with the XPS atomic per cent Ti concentration. Thus, the ToF-SIMS S(-), SO(-) and TiO(3)H(2)(-) intensities can be used to quantify the composition and amount of grafted polymer. ToF-SIMS also detected ions that were more characteristic of the polymer molecular structure (C(6)H(4)SO(3)(-) and C(8)H(7)SO(3)(-) from NaSS, C(4)H(5)O(2)(-) from MA), but the intensity of these peaks depended on the polymer thickness and composition. An in vitro cell culture test was carried out with human osteoblast-like cells to assess the influence of the grafted polymers on cell response. Cell adhesion after 30min of incubation showed significant differences between the grafted and ungrafted surfaces. The NaSS grafted surfaces showed the highest degree of cell adhesion while the MA-NaSS grafted surfaces showed the lowest degree of cell adhesion. After 4 weeks in vivo in rabbit femoral bones, bone was observed to be in direct contact with all implants. The percentage of mineralized tissue around the implants was similar for NaSS grafted and non-grafted implants (59% and 57%). The MA-NaSS grafted implant exhibited a lower amount of mineralized tissue (47%).

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.