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Below are abstracts to select publications relating to the Attana systems.

Posted with permission from Langmuir.

Langmuir, 23 (22), 11147 -11156, 2007

Mode of Heavy Meromyosin Adsorption and Motor Function Correlated with Surface Hydrophobicity and Charge

Nuria Albet-Torres, John O'Mahony, Christy Charlton, Martina Balaz, Patricia Lisboa, Teodor Aastrup, Alf Månsson, and Ian A. Nicholls

Abstract: 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 20-80^o). 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 (HMM^C) or via their positively charged N-terminal motor domain (HMM^N) 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 HMM^C configuration would be the dominant configuration at high contact angles but would be supplemented with electrostatically adsorbed HMM molecules (HMM^N configuration) at low contact angles. This would explain the higher initial HMM adsorption (from probability arguments) under the latter conditions. Furthermore, because the HMM^N 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.

Analytical Chemistry 79 (18): 6897-6902, Sep. 15, 2007

Photoderivatized Polymer Thin Films at Quartz Crystal Microbalance Surfaces: Sensors for Carbohydrate-Protein Interactions

Yuxin Pei, Hui Yu, Zhichao Pei, Matthias Theurer, Carolin Ammer, Sabine André, Hans-Joachim Gabius, Mingdi Yan and Olof Ramström

Abstract: Photoderivatized polymer-coated gold surfaces have been developed following a perfluorophenylazide-based double ligation strategy. Gold-plated quartz crystal microbalance (QCM) crystals were initially covalently functionalized with a monolayer of poly(ethylene glycol) (PEG), using photo- or thermolytic nitrene formation and insertion. The polymer surfaces were subsequently used as substrates for photoinsertion of carbohydrate-derivatized photoprobes, yielding different recognition motifs for selective protein binding. The resulting robust and biocompatible sensor surfaces were applied to a flow-through QCM instrument for monitoring lectin-carbohydrate interactions in real time. The results clearly show the predicted lectin selectivity, demonstrating the applicability of the approach.

Journal of Colloid and Interface Science, 313 (2), p.735-746, Sep 2007

Forces and friction between hydrophilic and hydrophobic surfaces: Influence of oleate species

Katarina Theander, Robert J. Pugh, Mark W. Rutland

Abstract: 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.

Biosensors and Bioelectronics 22 (2006) 42–48

Quartz crystal microbalance bioaffinity sensor for rapid identification of glycosyldisulfide lectin inhibitors from a dynamic combinatorial library

Zhichao Pei, Rikard Larsson, Teodor Aastrup, Henrik Anderson, Jean-Marie Lehn and Olof Ramström

Abstract: Carbohydrate–lectin interactions were probed with dynamic combinatorial libraries, using the plant lectin Concanavalin A as target species. The dynamic combinatorial libraries were generated from a pool of thiol components through reversible thiol–disulfide interchange, and screened using a simple and efficient method based on a quartz crystal microbalance setup. It was found that dimers based on 1-thio- and 6-thio-mannose analogues were the most active inhibitors. Furthermore, the results clearly show that the 6-thio-mannose possess unique characteristics compared to its oxygen-containing counterpart.

Protein Engineering, Design & Selection 2005 18(11):537-546

Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatorics

Kristina Jonas, Erhard Van Der Vries, Mikael T.I.Nilsson and Mikael Widersten

Abstract: New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins elected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.

Chemistry of Materials 17 (2005) 4092-4096

UV-Cross-Linked Poly(vinylpyridine) Thin Films as Reversibly Responsive Surfaces

Bernadette Harnish, Joshua T. Robinson, Zhichao Pei, Olof Ramström and Mingdi Yan

Abstract: 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.

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2707–2710

Redox-responsive and calcium-dependent switching of glycosyldisulfide interactions with Concanavalin A

Zhichao Pei, Teodor Aastrup, Henrik Anderson and Olof Ramström

Abstract: Glycosyldisulfides can interact efficiently with carbohydrate-binding entities. This has been shown for a range of thiosaccharide dimers when tested for their effects against the lectin Concanavalin A using a modified quartz crystal microbalance-technique. Contrary to the thiosaccharide monomers, showing no significant binding up to 10 mM, several of the dimers showed IC50-values in the low millimolar range. Three of the glycosyldisulfides tested also displayed very high positive apparent cooperativity effects that were found to be both calcium-dependent and redox-responsive.

Analytical Biochemistry 341 (2005) 89-93

Electro-immobilization of proinsulin C-peptide to a quartz crystal microbalance sensor chip for protein affinity purification

Ermias Melles, Henrik Anderson, Daniel Wallinder, Jawed Shafqat, Tomas Bergman, Teodor Aastrup and Hans Jörnvall

Abstract: Proinsulin C-peptide was electro-immobilized to a quartz crystal microbalance sensor chip, localizing this low-pI peptide for covalent attachment to activated surface carboxyl groups. The resulting chip was used in a continuous flow biosensor to capture anti-C-peptide antibodies, which could subsequently be eluted in 5% formic acid between air bubbles for efficient recovery and mass spectrometric identification. The method is reproducible through repeated cycles, providing affinity purification of proteins under real-time monitoring of the binding and elution processes.

Biosensors and Bioelectronics 21 (2005) 60–66

Study of real-time lectin–carbohydrate interactions on the surface of a quartz crystal microbalance

Zhichao Pei, Henrik Anderson, Teodor Aastrup and Olof Ramström

Abstract: A quartz crystal microbalance (QCM) biosensor system for lectin–carbohydrate interactions has been developed. Yeast mannan was immobilised on polystyrene-coated quartz crystals, and interactions tested with the lectin Concanavalin A (Con A). The biosensor could be easily operated, where mannan immobilisation and all binding analyses were performed in real-time using a flow-through system. The apparent binding constant for yeast mannan to Con A was estimated to be 0.4 _M, well in accordance to reported literature values. In addition, the effective concentration values (EC50-values) for a series of mannose/mannoside ligands, acting as competitors to the mannan/Con A interaction, were determined to range from 0.18 to 5.3 mM, in good correlation with a related enzyme-labeled lectin assay (ELLA) protocol.

Attana has prepared an Application Note on this work which can be downloaded here: Attana AppNote-Competition Study.pdf.

Analytica Chimica Acta 536 (2005) 191–196

Molecularly imprinted polymer thin films on quartz crystal microbalance using a surface bound photo-radical initiator

Theeraphon Piacham, Åsa Josell, Hans Arwin, Virapong Prachayasittikul and Lei Ye

Abstract: 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.