The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO(2) and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of similar to 6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems. (C) 2011 Elsevier Inc. All rights reserved.

Electrochemical (EC) quartz crystal microbalance with dissipation monitoring (ECQCM-D) is a new and powerful technique for the in situ study of adsorption phenomena. e.g., as a function of the potential of the substrate. When titanium Ti) is employed as the substrate, its oxidation behavior needs to be taken into account. Ti is always covered with a native oxide layer that can grow by, e.g., thermal oxidation or under anodic polarization. For biomolecular adsorption studies on oxidized Ti under applied potential, a stable oxide layer is desired in order to be able to distinguish the adsorption studies on oxidized Ti under applied potenital, a stable oxide layer is desired in order to be able to distinguish the adsorption phenomena and the oxide growth. Therefore, the oxidation of thermally evaporated Ti films was investigated in phosphate buffered saline by means of ECOCM-d, using a specially designed EC flow cell Upon stepping the potential applied to Ti up to 2.6 V vs standard hydrogen electrode (SHE), a fast increase of the mass was observed initially for each potential step evolving slowly to an asymptotic mass change after several hours. The oxide layer thickness increased as a quasi-linear function of the oxidation potential for potential up to 1.8 V vs SHE. The composition of the oxide layer was analyzed by X-ray photoelectron spectroscopy (XPS) it was mainly composed of TiO2 with a small percentage of suboxides (TiO and Ti2O3) primarily at the inner metal/oxide interface. The amount composed of TiO2, with a small percentage of suboxides TiO and Ti2O3 decreased with increasing oxidation potential. For each oxidation potential the calculated thickness obtained from ECQCM-D correlated well with the thickness obtained by XPS depth profiling. A procedure to prepare Ti samples with a stable oxide layer was successfully established for investigations on the influence of an electric field on the adsorption of biomolecules. As such, the effect of an applied potential on the adsorption behavior of lysozyme on oxidized Ti was investigated. It was observed that the adsorption of lysozyme on oxidized Ti was not influnced by the applied potential.

This article summarizes our most recent contributions to the rapidly growing field of supported lipid assemblies with emphasis on current studies addressing both fundamental and applied aspects of supported lipid bilayer (SLB) and tethered lipid vesicles (TLVs) to be utilized in sensing applications. The new insights obtained from combining the quartz crystal microbalance with dissipation monitoring technique with surface plasmon resonance are described, and we also present recent studies in which nanoplasmonic sensing has been used in studies of SLBs and TLVs. To gain full control over the spatial arrangement of TLVs in both two and three dimensions, we have developed a method for site-selective and sequence-specific sorting of DNA-tagged vesicles to surfaces modified with complementary DNA. The combination of this method with nanoplasmonic sensing formats is covered as well as the possibility of using DNA-modified vesicles for the detection of unlabeled DNA targets on the single-molecule level. Finally, a new method for membrane fusion induced by hybridization of vesicle-anchored DNA is demonstrated, including new results on content mixing obtained with vesicle populations encapsulating short, complementary DNA strands.

Self-assembled monolayers (SAMs) on gold exposing a-D-Galp-(1 ? 4)-ß-D-Galp-(1?4)-ß-D-Glcp (globotriose) are described. A synthetic pathway for the preparation of the bioactive carbohydrate globotriose coupled directly to bis(16-hydroxyhexadecanyl) disulfide ((HOC16H32S)2), as well as via tetra- or di(ethylene glycol) spacers, was developed. The SAMs were characterized by ellipsometry, contact angle goniometry, infrared reflection-absorption spectroscopy, and by their interactions with monoclonal antibodies. The ellipsometry measurements of mixed SAMs revealed thicknesses between 22 and 40 Å, depending on the ratio between carbohydrate and non-carbohydrate disulfides in the preparation solution. When the solution contained 10% or more of the carbohydrate adsorbate, the modified gold substrates displayed total wetting. Infrared reflection-absorption spectroscopy conferred well-ordered SAMs with a high degree of crystallinity. Furthermore, two monoclonal antibodies (IgM, MAHI 419 and IgG, MAHI 5) showed different affinity for mixed SAMs depending on the fraction and the structure of the carbohydrate component used (globotriose or globotriose tetra(ethylene glycol)), with the largest amount of protein bound for MAHI 419 at 1-10% of surface carbohydrate. These results demonstrate the usefulness of the SAM approach to explore molecular details such as the effect of a spacer or antigen distribution on antibody interactions at interfaces.

Temperature-programmed desorption (TPD) of self-assembled monolayers (SAMs) on gold is investigated by using in parallel mass spectrometry (MS) and infrared reflection-absorption spectroscopy (IRAS). Monolayers formed by HS(CH2)n-OH (n = 18, 22) and HS(CH2)15-CONH-(CH2CH2O)-H (EG1) are compared to reveal the influence of specifically introduced hydrogen-bonding groups on their thermal stability. The overall desorption process of the above molecules is found to occur in two main steps, a disordering of the alkyl chains followed by a complex series of decomposition/desorption reactions. The final step of the process involves desorption of sulfur from different chemisorption states. The amide-group-containing SAM, which is stabilized by lateral hydrogen bonds, displays a substantial delay of the alkyl chain disordering by about 50 K, as compared to the linear chain alcohols HS(CH2)n-OH. Moreover, the decomposition of the alkyls and the onset of sulfur desorption occur at a temperature that is higher by approximately 25 K as compared to the HS(CH2)18-OH SAM. The desorption process is also studied for two oligo(ethylene glycol)-terminated SAMs, HS(CH2)15-X-(CH2CH2O)4-H (EG4-SAMs), where X is -CONH- and -COO- linking groups. In addition to the molecular chain disordering, the decomposition/desorption process of the EG4-SAMs occurs in two steps. The first is associated with the loss of the oligomer portion and the second with the desorption of the alkylthiolate part of the molecule. Our study points out that lateral hydrogen bonding, introduced via amide groups, is a convenient way to improve the thermal stability of alkanthiolate SAMs.

The overall aim of this thesis has been to explore the use of self-assembled monolayers (SAMs) in model studies of biomolecular binding at interfaces. Molecular details of interfaces are difficult to address experimentally, and in spite of the fact that interactions in the living system commonly occur at interfaces, e.g. in the vicinity of a cell or a cell compartment, biomolecular interactions are generally studied in bulk solution. In special cases. such as in experiments with SAMs on metal surfaces, structural and functional information related to interfacial phenomena can be accessed more easily.

A strategy was developed for the synthesis of a series of oligo( ethylene glycol)-terminated alkanethiols (HS-(CH₂)m-CONH-(CH₂CH₂0)_n-H) with the objective of studying structure and stability of the corresponding SAMs on gold. Oligo(ethylene glycol) chains were coupled to alkyl chains via amide or ester linkages. Structural characterisation by infrared reflection-absorption spectroscopy (IRAS) revealed highly ordered SAMs. where the confonnation of the oligo(ethylene glycol) chains varied as a result of the intermolecular interactions in the monolayer. The SAMs were moderately hydrophilic (water contact angles ~30°). and protein adsorption studies indicated reasonable resistance toward simple protein systems. However. substantial amounts of proteins adsorbed from a more complex biofluid such as serum.

The trisaccharide globotriose is a recurrent structural motif of glycosphingolipids and is involved in various biological functions. According to IRAS studies, the SAMs of globotriose-terminated alkanethiols were found to form well-ordered structures. By surface plasmon resonance (SPR) biosensor experiment interactions of specific monoclonal antibodies with globotriose-containing SAMs proved sensitive to small fractions of carbohydrate (- 1%) in mixed SAMs. Different antibodies also responded distinctly different to a series of SAMs with increasing globotriose fractions. Thus. the SAM approach has great potential for studying of antibody binding to tailor-made interfaces, where molecular details such as spacer length and antigen distribution can be systematically varied.

Different immobilisations of globotriose-derived haptens to dextran-coated sensor chips in SPR biosensor experiments were investigated with the results being in qualitative agreement with solidphase immunoassays (ELISA). However. antibody interactions may be overlooked in the direct immobilisation of haptens to dextran. The work in this thesis suggests that these can be more easily detected using neoglycoproteins.

An SPR kinetic analysis of the interactions between carbonic anhydrase (CA) mutants and immobilised benzenesulphonamide inhibitors was performed. Dissociation rates were found to increase for a series of increasingly destabilised. point-mutated CA variants. The mutation was located far away from the binding site. suggesting that the observed effect on dissociation rate might be due to increased molecular dynamics.

A comparative study of the self-assembly and phase behavior of seven different oligo(ethylene glycol) (OEG)-terminated alkanethiols on polycrystalline gold surfaces is presented. The general structure of the compounds is HS(CH2)m-X-EGn, where m = 11, 15, n = 2, 4, 6, and the linkages X are amide (-CONH-), ester (-COO-), or ether (-O-) groups. The amide and ester groups give rise to the intermolecular hydrogen bonding and dipole-dipole interactions, respectively, whereas the ether lacks specific interactions. The results from contact angle goniometry, null ellipsometry, and infrared reflection-absorption spectroscopy (IRAS) indicate that the intermolecular interactions can be partly used to control the conformation and order of the OEG portion of the self-assembled monolayers (SAMs). It is shown that the lateral hydrogen bonding stabilizes the all-trans conformation of the EG4 tails in the SAMs. Further on, the mechanism behind the thermal phase behavior of the OEG SAMs is investigated using temperature-programmed IRAS in ultrahigh vacuum. In the present study we show that the earlier reported helix-to-all-trans conformational transition at 60°C in the SAM of HS(CH2)15CONH-EG6 (Valiokas, R., Östblom, M., Svedhem, S., Svensson, S. C. T., Liedberg, B. J. Phys. Chem. 2000, 104, 7565-7569.) is a result of the particular molecular design of the SAMs through the specifically built-in lateral hydrogen bonds. A shortening of the alkyl chain to 11 methylenes has no effect on the amide-EG6 phase behavior. Contrary, the ester- and ether- containing SAMs undergo a melting type of transitions at 52 and 68°C, respectively, similar to that observed for poly(ethylene glycol).

Low protein adsorption is believed advantageous for blood-contacting materials and ethylene glycols (EG)-based polymeric compounds are often attached to surfaces for this purpose. In the present study, the adsorption of fibrinogen, serum, and plasma were studied by ellipsometry on a series of well-defined oligo(EG) terminated alkane-thiols self-assembled on gold. The layers were prepared with compounds of the general structure HS-(CH2)15-CONH-EGn, where n = 2, 4, and 6. Methoxy-terminated tri(EG) undecanethiol and hydroxyl-terminated hexadecanethiol self-assembled monolayers (SAMs) were used as references. The results clearly demonstrate that the adsorption depends on the experimental conditions with small amounts of fibrinogen adsorbing from a single protein solution, but larger amounts of proteins from serum and plasma. The adsorption of fibrinogen and blood plasma decreased with an increasing number of EG repeats and was temperature-dependent. Significantly less serum adsorbed to methoxy tri(EG) than to hexa(EG) and more proteins remained on the latter surface after incubation in a sodium dodecyl sulfate (SDS) solution, indicating a looser protein binding to the methoxy-terminated surface. All surfaces adsorbed complement factor 3(C3) from serum and plasma, although no surface-mediated complement activation was observed. The present study points to the importance of a careful choice of the protein model system before general statements regarding the protein repellant properties of potential surfaces can be made.

The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s⁻¹. For the mutants, dissociation rates were faster (0.022–0.025 s⁻¹), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

A strategy for the synthesis of a series of closely related oligo(ethylene glycol)-terminated alkanethiol amides (principally HS(CH₂)_mCONH(CH₂CH₂O)_nH; m = 2, 5, 11, 15, n = 1, 2, 4, 6, 8, 10, 12) and analogous esters has been developed. These compounds were made to study the structure and stability of self-assembled monolayers (SAMs) on gold in the prospect of designing new biosensing interfaces. For this purpose, monodisperse heterofunctional oligo(ethylene glycols) with up to 12 units were prepared. Selective monoacylation of the symmetrical tetra- and hexa(ethylene glycol) diols as their mesylates with the use of silver(I) oxide was performed. The synthetic approach was based on carbodiimide couplings of various oligo(ethylene glycol) derivatives to ω-(acetylthio) carboxylic acids via a terminal amino or hydroxyl function. SAM structures on gold were studied with respect to thickness, wettability (water contact angles ∼30°), and conformation. A good fit was obtained for the relation between monolayer thickness (d) and the number of units in the oligo(ethylene glycol) chain (n):  d = 2.8n + 21.8 (Å). Interestingly, the corresponding infrared spectroscopy analysis showed a dramatic change in conformation of the oligomeric chains from all-trans (n = 4) to helical (n ≥ 6) conformation. A crystalline helical structure was observed in the SAMs for n > 6.