Publications: Surface Chemistry

Hyaluronan (HA), a polymeric glycosaminoglycan ubiquitously present in higher animals is hydrolyzed by hyaluronidases (HAases). Here we used bee HAase as a model enzyme to study HA-HAase interaction. Located in close proximity to the active center, a bulky surface loop, which appears to obstruct one end of the substrate binding groove, was found to be functionally involved in HA turnover. To better apprehend kinetic changes in substrate interaction, binding of high molecular weight HA to catalytically inactive HAase was monitored by means of Quartz Crystal Microbalance technology. Replacement of the delimiting loop by a tetrapeptide interconnection increased the affinity for HA up to 100-fold, with a KD below 1 nM being the highest affinity amongst HA-binding proteins surveyed so far. The experimental data of HA-HAase interaction were further validated showing best fit to the theoretically proposed sequential twosite model. Besides the one, which had been shown previously in course of X-ray structure determination, a previously unrecognized binding site works in conjunction with an unbinding loop that facilitates liberation of hydrolyzed HA.

© The American Society for Biochemistry and Molecular Biology 2011

Collagen, the major component of extracellular matrix (ECM) and the most abundant protein in the human body, is implicated in the development of atherosclerosis. Collagen types I and III were immobilized on fused-silica capillary to investigate their shape, size and structure by atomic force microscopy (AFM). For comparison, collagen was also immobilized on a mica surface. Our studies demonstrated that not only does the adsorption pattern on the substrate vary with the type of collagen, but also the substrate material plays an important role in the fibril formation process. Decorin, which promotes the binding of low-density lipoprotein (LDL) particles with collagen, was investigated for its effect on the fibrillogenesis. On both substrate materials, addition of decorin clearly reduced the fibril diameter of collagen surfaces. Moreover, a quartz crystal microbalance (QCM)-based biosensor approach was applied to clarify and evaluate the affinity of different collagen coatings immobilized on a silicon dioxide sensor chip toward apolipoprotein B-100, the major protein of LDL. The results confirmed the importance of collagen type and their fibrillogenesis on the binding of the positive residues of apolipoprotein B-100 on negatively charged collagen surfaces.

© Royal Society of Chemistry 2011

A series of streptavidin-mimicking molecularly imprinted polymers has been developed and evaluated for their biotin binding characteristics. A combination of molecular dynamics and NMR spectroscopy was used to examine potential polymer systems, in particular with the functional monomers methacrylic acid and 2-acrylamidopyridine. The synthesis of copolymers of ethylene dimethacrylate and one or both of these functional monomers was performed. A combination of radioligand binding studies and surface area analyses demonstrated the presence of selectivity in polymers prepared using methacrylic acid as the functional monomer. This was predicted by the molecular dynamics studies showing the power of this methodology as a prognostic tool for predicting the behavior of molecularly imprinted polymers.

© Springer-Verlag 2011

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pKa, antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl 3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pKa is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab2 antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

© 2009 Elsevier Inc.

Dynamic acrylamide/acrylate polymeric brushes were synthesized at gold-plated quartz crystal surfaces. The crystals were initially coated with polystyrene-type thin films, derivatized with photolabile iniferter groups, and subsequently subjected to photoinitiated polymerization in acrylamide/acrylate monomer feeds. This surface-confined polymerization method enabled direct photocontrol over the polymerization, as followed by increased frequency responses of the crystal oscillations in a quartz crystal microbalance (QCM) instrumentation. The produced polymer layers were also found to be highly sensitive to external acid/base stimuli. Large oscillation frequency shifts were detected when the brushes were exposed to buffer solutions of different pH. The dynamic behavior of the resulting polymeric brushes was evaluated and the extent of expansion and contraction of the films monitored by the QCM setup in situ in real-time. The resulting responses were rapid and the effects were fully reversible. Low pH resulted in full contractions of the films, whereas higher pH yielded maximal expansion in order to minimize repulsion around the charged acrylate centers. The surfaces proved also highly robust, since the responsiveness was reproducible over many cycles of repeated expansion and contraction.

© 2008 American Chemical Society

The atomic force microscope has been used to investigate normal surface forces and lateral friction forces at different concentrations of sodium oleate, a frequently used fatty acid in the deinking process. The measurements have been performed using the colloidal probe technique with bead materials consisting of cellulose and silica. Cellulose was used together with a printing ink alkyd resin and mica, whereas silica was used with a hydrophobized silica wafer. The cellulose–alkyd resin system showed stronger double layer repulsion and the friction was reduced with increasing surfactant concentration. The adhesive interaction disappeared immediately on addition of sodium oleate. The normal surface forces for cellulose–mica indicated no apparent adsorption of the sodium oleate however, the friction coefficient increased on addition of sodium oleate, which we ascribe to some limited adsorption increasing the effective surface roughness. The silica–hydrophobic silica system showed a completely different surface force behavior at the different concentrations. An attractive hydrophobic interaction was evident since the surfaces jumped into adhesive contact at a longer distance than the van der Waals forces would predict. The strong adhesion was reflected in the friction forces as a nonlinear relationship between load and friction and a large friction response at zero applied load. Indirect evidence of adsorption to the hydrophilic silica surface was also observed in this case, and QCM studies were performed to confirm the adsorption of material to both surfaces.

© 2007 Published by Elsevier Inc.

We present a versatile chamber (“atmospheric corrosion cell”) for soft x-ray absorption/emission spectroscopy of metal surfaces in a corrosive atmosphere allowing novel in situ electronic structure studies. Synchrotron x rays passing through a thin window separating the corrosion cell interior from a beamline vacuum chamber probe a metal film deposited on a quartz crystal microbalance (QCM) or on the inside of the window.We present some initial results on chloride induced corrosion of iron surfaces in humidified synthetic air. By simultaneous recording of QCM signal and soft x-ray emission from the corroding sample, correlation between mass changes and variations in spectral features is facilitated.

© 2007 American Institute of Physics.

The influence of flow cell geometry on sample dispersion in a quartz crystal microbalance (QCM) biosensor system was investigated. A circular and a rectangular flow cell and corresponding sensor electrodes were studied experimentally and modelled using a coupled Navier–Stokes and convection–diffusion model. Finite element simulations showed that dispersion phenomena in a flow cell can be significantly reduced with the rectangular flow cell compared to a circular system. Experimental results from measurement of the time-dependent viscosity change of a model sample indicate that the sample delivery system has a predominant effect on the dispersion of the whole sensor system. Consequently, improvement of the sensor flow cell should be accompanied with improvement of the sample delivery system. With reference to kinetic studies of biological interactions, the current dispersion should have little effect on the results for studies of interaction pairs with relatively slow to normal binding rates such as antibody–antigen interactions. Incentive for further development of the flow cell and sample delivery system exists primarily for applications with high reaction rates such as for certain receptor ligand interactions.

© 2007 Elsevier B.V. All rights reserved.

This paper investigates a novel quartz crystal microbalance (QCM) biosensor with a small and rectangular flow cell along with a correspondingly shaped crystal electrode. The sensor was evaluated with impedance analysis and compared to standard circular sensor crystals and sensor crystals with small circular electrodes. Comparative QCM measurements on an antibody–antigen interaction system were carried out on the rectangular and standard circular sensor systems. Impedance analysis and subsequent data extraction of the three different sensor crystals showed that the smaller sensors had significantly higher Q-values in air, but that liquid load on the electrodes lowered the Q-values radically for all crystals. Under liquid load, Q-values for the standard circular and the rectangular sensors were similar whereas the Q-value for the small circular sensor was 50% higher. QCM experiments showed that the QCM system with rectangular crystal electrodes was fully functional in a liquid environment. The rectangular system showed higher and more rapid responses for series of antibody injections, albeit at a higher noise level than the standard system. The study elucidates a significant potential for improvement of sensor performance by optimising the sensor electrode size and shape together with the flow cell geometry.

© 2007 Elsevier B.V. All rights reserved.

The in vitro motility assay is valuable for fundamental studies of actomyosin function and has recently been combined with nanostructuring techniques for the development of nanotechnological applications. However, the limited understanding of the interaction mechanisms between myosin motor fragments (heavy meromyosin, HMM) and artificial surfaces hampers the development as well as the interpretation of fundamental studies. Here we elucidate the HMM-surface interaction mechanisms for a range of negatively charged surfaces (silanized glass and SiO₂), which is relevant both to nanotechnology and fundamental studies. The results show that the HMM-propelled actin filament sliding speed (after a single injection of HMM, 120μg/mL) increased with the contact angle of the surfaces (in the range of 20-80o). However, quartz crystal microbalance (QCM) studies suggested a reduction in the adsorption of HMM (with coupled water) under these conditions. This result and actin filament binding data, together with previous measurements of the HMM density (Sundberg, M.; Balaz, M.; Bunk, R.; Rosengren-Holmberg, J. P.; Montelius, L.; Nicholls, I. A.; Omling, P.; Tågerud, S.; Månsson, A. Langmuir 2006, 22, 7302-7312. Balaz, M.; Sundberg, M.; Persson, M.; Kvassman, J.; Månsson, A. Biochemistry 2007, 46, 7233-7251), are consistent with (1) an HMM monolayer and (2) different HMM configurations at different contact angles of the surface. More specifically, the QCM and in vitro motility assay data are consistent with a model where the molecules are adsorbed either via their flexible C-terminal tail part (HMMC) or via their positively charged N-terminal motor domain (HMMN) without other surface contact points. Measurements of ξ potentials suggest that an increased contact angle is correlated with a reduced negative charge of the surfaces. As a consequence, the HMMC configuration would be the dominant configuration at high contact angles but would be supplemented with electrostatically adsorbed HMM molecules (HMMN configuration) at low contact angles. This would explain the higher initial HMM adsorption (from probability arguments) under the latter conditions. Furthermore, because the HMMN; mode would have no actin binding it would also account for the lower sliding velocity at low contact angles. The results are compared to previous studies of the microtubule-kinesin system and are also discussed in relation to fundamental studies of actomyosin and nanotechnological developments and applications.

© 2007 American Chemical Society. All rights reserved.

Cover image posted with permission from Langmuir.

We report that UV-cross-linked poly(4-vinylpyridine) (P4VP) films acted as reversibly responsive coatings that controlled surface wettability and swelling toward external stimuli: solvent and pH. The polymer films were prepared simply by spin-coating a solution of P4VP followed by UV irradiation. These cross-linked films, when treated with chloroform, showed 31% increase in film thickness whereas films extracted with methylene chloride or n-butanol exhibited a slight decrease. The increase in film thickness was due to the protonation of pyridyl groups by hydrogen chloride resulting from the photodegeneration of chloroform. The film expanded to minimize repulsion around the charged centers. This hypothesis was further confirmed by exposing the cross-linked film to hydrogen chloride vapor. The film expanded 37% whereas no thickness increase was observed for films exposed to ammonia or methanol vapors. The extent of swelling was monitored in situ using a quartz crystal microbalance sensor. Large oscillation frequency shifts were detected when the UV-cross-linked P4VP film was exposed to acidic buffer solutions. The changes were rapid, and the effect was reversible.

© 2005 American Chemical Society. All rights reserved.

The present work aims to study preparation of thin MIP films on a gold-coated QCM resonator in a well-controlled and reproducible manner. A surface-bound photo-radical initiator was used to furnish an imprinting polymerization in a dilute solution of template, functional monomer and crosslinker. Prior to polymerization, the photo-initiator was covalently coupled to a self-assembled monolayer of carboxyl terminated alkanethiol on a gold surface. With this approach we were able to control the thickness of the MIP film to be below 50 nm, where the selective recognition of target analytes can be easily detected by the underlying quartz crystal resonator. When used in a flow injection analysis system, the assembled QCM sensor generated a large frequency change (>30 Hz) upon encountering a small amount of analyte (0.19 mM). The sensor had a very short response time (<1 min), and displayed certain chiral selectivity towards the original template, (S)-propranolol at a concentration higher than 0.38mM in aqueous solution.

© 2004 Elsevier B.V. All rights reserved.