The use of heterogeneous chemistry for the characterization of functional groups at the gas/particle interface of soot and TiO(2) nanoparticles

Six gases (N((CH3)3), NH2OH, CF3COOH, HCl, NO2, O3) were selected to probe the surface of seven combustion aerosol (amorphous carbon, flame soot) and three types of TiO2 nanoparticles using heterogeneous, that is gas-surface reactions. The gas uptake to saturation of the probes was measured under molecular flow conditions in a Knudsen flow reactor and expressed as a density of surface functional groups on a particular aerosol, namely acidic (carboxylic) and basic (conjugated oxides such as pyrones, N-heterocycles) sites, carbonyl (R1-C(O)-R2) and oxidizable (olefinic, -OH) groups. The limit of detection was generally well below 1% of a formal monolayer of adsorbed probe gas. With few exceptions most investigated aerosol samples interacted with all probe gases which points to the coexistence of different functional groups on the same aerosol surface such as acidic and basic groups. Generally, the carbonaceous particles displayed significant differences in surface group density: Printex 60 amorphous carbon had the lowest density of surface functional groups throughout, whereas Diesel soot recovered from a Diesel particulate filter had the largest. The presence of basic oxides on carbonaceous aerosol particles was inferred from the ratio of uptakes of CF3COOH and HCl owing to the larger stability of the acetate compared to the chloride counterion in the resulting pyrylium salt. Both soots generated from a rich and a lean hexane diffusion flame had a large density of oxidizable groups similar to amorphous carbon FS 101. TiO2 15 had the lowest density of functional groups among the three studied TiO2 nanoparticles for all probe gases despite the smallest size of its primary particles. The used technique enabled the measurement of the uptake probability of the probe gases on the various supported aerosol samples. The initial uptake probability, g0, of the probe gas onto the supported nanoparticles differed significantly among the various investigated aerosol samples but was roughly correlated with the density of surface groups, as expected. [Authors]

A fluorescent peptide substrate for the surface metalloprotease of Leishmania.

A fluorescent oligopeptide substrate for the promastigote surface protease (PSP) of Leishmania was designed using the data reported for the substrate specificity of the enzyme (Bouvier, J., Schneider, P., Etges, R. J., and Bordier, C. 1990. Biochemistry 29, 10113-10119). The indole fluorescence of the tryptophan residue was efficiently quenched through resonance energy transfer by an N-terminal dansyl group located five amino acid residues away. The heptapeptide, dansyl-A-Y-L-K-K-W-V-NH2, was cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 8.8 x 10(6) M-1sec-1. Hydrolysis by the enzyme results in a time-dependent increase of fluorescence intensity of 3.7-fold. Assays can be designed based on the tryptophan fluorescence at 360 nm or by individual product analyses using thin-layer chromatography. The synthetic substrate is readily cleaved by the metalloprotease at the surface of fixed promastigotes. The specificity and sensitivity of such internally quenched fluorescent peptide substrate will facilitate the identification of novel inhibitors for the enzyme and aid in detailed studies on its enzymology.

The C-terminal domain of Plasmodium falciparum merozoite surface protein 3 self-assembles into alpha-helical coiled coil tetramer.

Proteins located on the surface of the pathogenic malaria parasite Plasmodium falciparum are objects of intensive studies due to their important role in the invasion of human cells and the accessibility to host antibodies thus making these proteins attractive vaccine candidates. One of these proteins, merozoite surface protein 3 (MSP3) represents a leading component among vaccine candidates; however, little is known about its structure and function. Our biophysical studies suggest that the 40 residue C-terminal domain of MSP3 protein self-assembles into a four-stranded alpha-helical coiled coil structure where alpha-helices are packed "side-by-side". A bioinformatics analysis provides an extended list of known and putative proteins from different species of Plasmodium which have such MSP3-like C-terminal domains. This finding allowed us to extend some conclusions of our studies to a larger group of the malaria surface proteins. Possible structural and functional roles of these highly conserved oligomerization domains in the intact merozoite surface proteins are discussed.

Chemical probes that differentially modulate peroxisome proliferator-activated receptor alpha and BLTR, nuclear and cell surface receptors for leukotriene B(4).

Peroxisome proliferator-activated receptor alpha (PPARalpha)is a nuclear receptor for various fatty acids, eicosanoids, and hypolipidemic drugs. In the presence of ligand, this transcription factor increases expression of target genes that are primarily associated with lipid homeostasis. We have previously reported PPARalpha as a nuclear receptor of the inflammatory mediator leukotriene B(4) (LTB(4)) and demonstrated an anti-inflammatory function for PPARalpha in vivo (Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39-43). LTB(4) also has a cell surface receptor (BLTR) that mediates proinflammatory events, such as chemotaxis and chemokinesis (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624). In this study, we report on chemical probes that differentially modulate activity of these two LTB(4) receptors. The compounds selected were originally characterized as synthetic BLTR effectors, both agonists and antagonists. Here, we evaluate the compounds as effectors of the three PPAR isotypes (alpha, beta, and gamma) by transient transfection assays and also determine whether the compounds are ligands for these nuclear receptors by coactivator-dependent receptor ligand interaction assay, a semifunctional in vitro assay. Because the compounds are PPARalpha selective, we further analyze their potency in a biological assay for the PPARalpha-mediated activity of lipid accumulation. These chemical probes will prove invaluable in dissecting processes that involve nuclear and cell surface LTB(4) receptors and also aid in drug discovery programs.

Surface-functionalized ultrasmall superparamagnetic nanoparticles as magnetic delivery vectors for camptothecin.

Drug-nanoparticle conjugates: The anticancer drug camptothecin (CPT) was covalently linked at the surface of ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) via a linker, allowing drug release by cellular esterases. Nanoparticles were hierarchically built to achieve magnetically-enhanced drug delivery to human cancer cells and antiproliferative activity.The linking of therapeutic drugs to ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) allowing intracellular release of the active drug via cell-specific mechanisms would achieve tumor-selective magnetically-enhanced drug delivery. To validate this concept, we covalently attached the anticancer drug camptothecin (CPT) to biocompatible USPIOs (iron oxide core, 9-10 nm; hydrodynamic diameter, 52 nm) coated with polyvinylalcohol/polyvinylamine (PVA/aminoPVA). A bifunctional, end-differentiated dicarboxylic acid linker allowed the attachment of CPT to the aminoPVA as a biologically labile ester substrate for cellular esterases at one end, and as an amide at the other end. These CPT-USPIO conjugates exhibited antiproliferative activity in vitro against human melanoma cells. The intracellular localization of CPT-USPIOs was confirmed by transmission electron microscopy (iron oxide core), suggesting localization in lipid vesicles, and by fluorescence microscopy (CPT). An external static magnetic field applied during exposure increased melanoma cell uptake of the CPT-USPIOs.

Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering.

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.

Surface modification of biomaterials for conjugation with human fetal osteoblasts.

Surface functionalization of hydroxyapatite (HA) and beta-tricalcium phosphate (TCP) bioceramics with chemical ligands containing a pyrrogallol moiety was developed to improve the adhesion of bone cell precursors to the biomaterials. Fast and biocompatible copper-free click reaction with azido-modified human fetal osteoblasts resulted in improved cell binding to both HA and TCP bioceramics, opening the way for using this methodology in the preparation of cell-engineered bone implants.

Understanding and modulating the competitive surface-adsorption of proteins through coarse-grained molecular dynamics simulations

It is now well accepted that cellular responses to materials in a biological medium reflect greatly the adsorbed biomolecular layer, rather than the material itself. Here, we study by molecular dynamics simulations the competitive protein adsorption on a surface (Vroman effect), i.e. the non-monotonic behavior of the amount of protein adsorbed on a surface in contact with plasma as functions of contact time and plasma concentration. We find a complex behavior, with regimes during which small and large proteins are not necessarily competing between them, but are both competing with others in solution ("cooperative" adsorption). We show how the Vroman effect can be understood, controlled and inverted.

Anthracene-tethered ruthenium(II) arene complexes as tools to visualize the cellular localization of putative organometallic anticancer compounds.

Anthracene derivatives of ruthenium(II) arene compounds with 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (pta) or a sugar phosphite ligand, viz., 3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-d-glucofuranoside, were prepared in order to evaluate their anticancer properties compared to the parent compounds and to use them as models for intracellular visualization by fluorescence microscopy. Similar IC(50) values were obtained in cell proliferation assays, and similar levels of uptake and accumulation were also established. The X-ray structure of [{Ru(η(6)-C(6)H(5)CH(2)NHCO-anthracene)Cl(2)(pta)] is also reported.

Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation.

Solid tumor growth triggers a wound healing response. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts (also referred to as cancer-associated fibroblasts) primarily, but not exclusively, in response to transforming growth factor-β (TGF-β). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among proteases implicated in stroma remodeling, matrix metalloproteinases (MMPs), including MMP-9, play a prominent role. Recent evidence indicates that MMP-9 recruitment to the tumor cell surface enhances tumor growth and invasion. In the present work, we addressed the potential relevance of MMP-9 recruitment to and activity at the surface of fibroblasts. We show that recruitment of MMP-9 to the fibroblast cell surface occurs through its fibronectin-like (FN) domain and that the molecule responsible for the recruitment is lysyl hydroxylase 3 (LH3). Functional assays suggest that both pro- and active MMP-9 trigger α-smooth muscle actin expression in cultured fibroblasts, reflecting myofibroblast differentiation, possibly as a result of TGF-β activation. Moreover, the recombinant FN domain inhibited both MMP-9-induced TGF-β activation and α-smooth muscle actin expression by displacing MMP-9 from the fibroblast cell surface. Together our results uncover LH3 as a new docking receptor of MMP-9 on the fibroblast cell surface and demonstrate that the MMP-9 FN domain is essential for the interaction. They also show that the recombinant FN domain inhibits MMP-9-induced TGF-β activation and fibroblast differentiation, providing a potentially attractive therapeutic reagent toward attenuating tumor progression where MMP-9 activity is strongly implicated.

Label-free detection of tobramycin in serum by transmission-localized surface plasmon resonance.

In order to improve the efficacy and safety of treatments, drug dosage needs to be adjusted to the actual needs of each patient in a truly personalized medicine approach. Key for widespread dosage adjustment is the availability of point-of-care devices able to measure plasma drug concentration in a simple, automated, and cost-effective fashion. In the present work, we introduce and test a portable, palm-sized transmission-localized surface plasmon resonance (T-LSPR) setup, comprised of off-the-shelf components and coupled with DNA-based aptamers specific to the antibiotic tobramycin (467 Da). The core of the T-LSPR setup are aptamer-functionalized gold nanoislands (NIs) deposited on a glass slide covered with fluorine-doped tin oxide (FTO), which acts as a biosensor. The gold NIs exhibit localized plasmon resonance in the visible range matching the sensitivity of the complementary metal oxide semiconductor (CMOS) image sensor employed as a light detector. The combination of gold NIs on the FTO substrate, causing NIs size and pattern irregularity, might reduce the overall sensitivity but confers extremely high stability in high-ionic solutions, allowing it to withstand numerous regeneration cycles without sensing losses. With this rather simple T-LSPR setup, we show real-time label-free detection of tobramycin in buffer, measuring concentrations down to 0.5 μM. We determined an affinity constant of the aptamer-tobramycin pair consistent with the value obtained using a commercial propagating-wave based SPR. Moreover, our label-free system can detect tobramycin in filtered undiluted blood serum, measuring concentrations down to 10 μM with a theoretical detection limit of 3.4 μM. While the association signal of tobramycin onto the aptamer is masked by the serum injection, the quantification of the captured tobramycin is possible during the dissociation phase and leads to a linear calibration curve for the concentrations over the tested range (10-80 μM). The plasmon shift following surface binding is calculated in terms of both plasmon peak location and hue, with the latter allowing faster data elaboration and real-time display of the results. The presented T-LSPR system shows for the first time label-free direct detection and quantification of a small molecule in the complex matrix of filtered undiluted blood serum. Its uncomplicated construction and compact size, together with the remarkable performances, represent a leap forward toward effective point-of-care devices for therapeutic drug concentration monitoring.

Surface Studies of Thiolate Adsorbates on Some Metals

The results and discussions in this thesis are based on my studies about selfassembled thiol layers on gold, platinum, silver and copper surfaces. These kinds of layers are two-dimensional, one molecule thick and covalently organized at the surface. They are an easy way to modify surface properties. Self-assembly is today an intensive research field because of the promise it holds for producing new technology at nanoscale, the scale of atoms and molecules. These kinds of films have applications for example, in the fields of physics, biology, engineering, chemistry and computer science. Compared to the extensive literature concerning self-assembled monolayers (SAMs) on gold, little is known about the structure and properties of thiolbased SAMs on other metals. In this thesis I have focused on thiol layers on gold, platinum, silver and copper substrates. These studies can be regarded as a basic study of SAMs. Nevertheless, an understanding of the physical and chemical nature of SAMs allows the correlation between atomic structure and macroscopic properties. The results can be used as a starting point for many practical applications. X-ray photoelectron spectroscopy (XPS) and synchrotron radiation excited high resolution photoelectron spectroscopy (HR-XPS) together with time-offlight secondary ion mass spectrometry (ToF-SIMS) were applied to investigate thin organic films formed by the spontaneous adsorption of molecules on metal surfaces. Photoelectron spectroscopy was the main method used in these studies. In photoelectron spectroscopy, the sample is irradiated with photons and emitted photoelectrons are energy-analyzed. The obtained spectra give information about the atomic composition of the surface and about the chemical state of the detected elements. It is widely used in the study of thin layers and is a very powerful tool for this purpose. Some XPS results were complemented with ToF-SIMS measurements. It provides information on the chemical composition and molecular structure of the samples. Thiol (1-Dodecanethiol, CH3(CH2)11SH) solution was used to create SAMs on metal substrates. Uniform layers were formed on most of the studied metal surfaces. On platinum, surface aligned molecules were also detected in investigations by XPS and ToF-SIMS. The influence of radiation on the layer structure was studied, leading to the conclusion that parts of the hydrocarbon chains break off due to radiation and the rest of the layer is deformed. The results obtained showed differences depending on the substrate material. The influence of oxygen on layer formation was also studied. Thiol molecules were found to replace some of the oxygen from the metal surfaces.

Influence of plasma modification on surface properties and offset printability of coated paper

The properties of the paper surface play a crucial role in ensuring suitable quality and runnability in various converting and finishing operations, such as printing. Plasma surface modification makes it possible to modify the surface chemistry of paper without altering the bulk material properties. This also makes it possible to investigate the role of the surface chemistry alone on printability without influencing the porous structure of the pigment-coated paper. Since the porous structure of a pigment coating controls both ink setting and optical properties, surface chemical changes created by a plasma modification have a potential to decouple these two effects and to permit a better optimization of them both. The aim of this work was to understand the effects of plasma surface modification on paper properties, and how it influences printability in the sheet-fed offset process. The objective was to broaden the fundamental understanding of the role of surface chemistry on offset printing. The effects of changing the hydrophilicity/ hydrophobicity and the surface chemical composition by plasma activation and plasma coatings on the properties of coated paper and on ink-paper interactions as well as on sheet-fed offset print quality were investigated. In addition, the durability of the plasma surface modification was studied. Nowadays, a typical sheet-fed offset press also contains units for surface finishing, for example UVvarnishing. The role of the surface chemistry on the UV-varnish absorption into highly permeable and porous pigment-coated paper was also investigated. With plasma activation it was possible to increase the surface energy and hydrophilicity of paper. Both polar and dispersion interactions were found to increase, although the change was greater in the polar interactions due to induced oxygen molecular groups. The results indicated that plasma activation takes place particularly in high molecular weight components such as the dispersion chemicals used to stabilize the pigment and latex particles. Surface composition, such as pigment and binder type, was found to influence the response to the plasma activation. The general trend was that pilot-scale treatment modified the surface chemistry without altering the physical coating structure, whereas excessive laboratory-scale treatment increased the surface roughness and reduced the surface strength, which led to micro-picking in printing. It was shown that pilot-scale plasma activation in combination with appropriate ink oils makes it possible to adjust the ink-setting rate. The ink-setting rate decreased with linseed-oil-based inks, probably due to increased acid-base interactions between the polar groups in the oil and the plasma-treated paper surface. With mineral-oil-based inks, the ink setting accelerated due to plasma activation. Hydrophobic plasma coatings were able to reduce or even prevent the absorption of dampening water into pigmentcoated paper, even when the dampening water was applied under the influence of nip pressure. A uniform hydrophobic plasma coating with sufficient chemical affinity with ink gave an improved print quality in terms of higher print density and lower print mottle. It was also shown that a fluorocarbon plasma coating reduced the free wetting of the UV-varnish into the highly permeable and porous pigment coating. However, when the UV-varnish was applied under the influence of nip pressure, which leads to forced wetting, the role of the surface chemical composition seems to be much less. A decay in surface energy and wettability occurred during the first weeks of storage after plasma activation, after which it leveled off. However, the oxygen/carbon elemental ratio did not decrease as a function of time, indicating that ageing could be caused by a re-orientation of polar groups or by a contamination of the surface. The plasma coatings appeared to be more stable when the hydrophobicity was higher, probably due to fewer interactions with oxygen and water vapor in the air.

Surface characterization of chemically modified fiber, wood and paper

Inorganic-organic sol-gel hybrid coatings can be used for improving and modifying properties of wood-based materials. By selecting a proper precursor, wood can be made water repellent, decay-, moisture- or UV-resistant. However, to control the barrier properties of sol-gel coatings on wood substrates against moisture uptake and weathering, an understanding of the surface morphology and chemistry of the deposited sol-gel coatings on wood substrates is needed. Mechanical pulp is used in production of wood-containing printing papers. The physical and chemical fiber surface characteristics, as created in the chosen mechanical pulp manufacturing process, play a key role in controlling the properties of the end-use product. A detailed understanding of how process parameters influence fiber surfaces can help improving cost-effectiveness of pulp and paper production. The current work focuses on physico-chemical characterization of modified wood-based materials with surface sensitive analytical tools. The overall objectives were, through advanced microscopy and chemical analysis techniques, (i) to collect versatile information about the surface structures of Norway spruce thermomechanical pulp fiber walls and understand how they are influenced by the selected chemical treatments, and (ii) to clarify the effect of various sol-gel coatings on surface structural and chemical properties of wood-based substrates. A special emphasis was on understanding the effect of sol-gel coatings on the water repellency of modified wood and paper surfaces. In the first part of the work, effects of chemical treatment on micro- and nano-scale surface structure of 1st stage TMP latewood fibers from Norway spruce were investigated. The chemicals applied were buffered sodium oxalate and hydrochloric acid. The outer and the inner fiber wall layers of the untreated and chemically treated fibers were separately analyzed by light microscopy, atomic force microscopy and field-emission scanning electron microscopy. The selected characterization methods enabled the demonstration of the effect of different treatments on the fiber surface structure, both visually and quantitatively. The outer fiber wall areas appeared as intact bands surrounding the fiber and they were clearly rougher than areas of exposed inner fiber wall. The roughness of the outer fiber wall areas increased most in the sodium oxalate treatment. The results indicated formation of more surface pores on the exposed inner fiber wall areas than on the corresponding outer fiber wall areas as a result of the chemical treatments. The hydrochloric acid treatment seemed to increase the surface porosity of the inner wall areas. In the second part of the work, three silane-based sol-gel hybrid coatings were selected in order to improve moisture resistance of wood and paper substrates. The coatings differed from each other in terms of having different alkyl (CH3–, CH3-(CH2)7–) and fluorocarbon (CF3–) chains attached to the trialkoxysilane sol-gel precursor. The sol-gel coatings were deposited by a wet coating method, i.e. spraying or spreading by brush. The effect of solgel coatings on surface structural and chemical properties of wood-based substrates was studied by using advanced surface analyzing tools: atomic force microscopy, X-ray photoelectron spectroscopy and time-of-flight secondary ion spectroscopy. The results show that the applied sol-gel coatings, deposited as thin films or particulate coatings, have different effects on surface characteristics of wood and wood-based materials. The coating which has a long hydrocarbon chain (CH3-(CH2)7–) attached to the silane backbone (octyltriethoxysilane) produced the highest hydrophobicity for wood and wood-based materials.

DNA-lipid systems: A physical chemistry study

It is well known that the interaction of polyelectrolytes with oppositely charged surfactants leads to an associative phase separation; however, the phase behavior of DNA and oppositely charged surfactants is more strongly associative than observed in other systems. A precipitate is formed with very low amounts of surfactant and DNA. DNA compaction is a general phenomenon in the presence of multivalent ions and positively charged surfaces; because of the high charge density there are strong attractive ion correlation effects. Techniques like phase diagram determinations, fluorescence microscopy, and ellipsometry were used to study these systems. The interaction between DNA and catanionic mixtures (i.e., mixtures of cationic and anionic surfactants) was also investigated. We observed that DNA compacts and adsorbs onto the surface of positively charged vesicles, and that the addition of an anionic surfactant can release DNA back into solution from a compact globular complex between DNA and the cationic surfactant. Finally, DNA interactions with polycations, chitosans with different chain lengths, were studied by fluorescence microscopy, in vivo transfection assays and cryogenic transmission electron microscopy. The general conclusion is that a chitosan effective in promoting compaction is also efficient in transfection.