Artificial receptors for membrane glycoproteins

Bei Wheat germ agglutinin (WGA) handelt es sich um ein Pflanzenlektin, das eine grundlegende Rolle in der Biotechnologie und er Biosensorik spielt, weil es mit Viren und Zellen im Zuge von Infektionsvorgängen ebenso reagiert, wie mit Oligosaccharieden, die auf den Zelloberflächen verschiedener Organismen vorhanden sind. Daher ist WGA auch interessanter Analyt für massensensitive Messungen, die Einblicke in die Erkennung und die ihr zugrundeliegenden Wechselwirkungen gestatten. Im Zuge dieser Arbeit wurden zwei verschiedene Rezeptorstrategien zur Sensorik von WGA mittels Quarzmikrowaage (quartz crystal microbalance – QCM) entwickelt, nämlich die Immobilisierung glykosidischer Rezeptoranaloga sowie molekular geprägte Polymer (molecularly imprinted polymers – MIP). Die entsprechenden Oberflächen wurden auch mittels Rastertunnelmikroskopie charakterisiert. Da diese Technik leitende Oberflächen benötigt, wurden auf die MIP Goldschichten durch Sputtering aufgebracht. Dadurch war es möglich tatsächlich Kavitäten in der Größe von WGA-Dimeren auf der Oberfläche zu visualisieren. Als Rezeptoranalogon wurde mit p-Nitrophenol und dann Cystein modifiziertes N-Acetyl-D-Glucosamin verwendet und über die SH-Gruppe des Cysteins auf Goldoberflächen immobilisiert. Aufgrund der Größe der Analytmoleküle erwies sich die Beschichtung mit einer kompletten Monolage des Rezeptors nicht als zielführend, da die Bindung dann sterisch behindert wurde. Deswegen mußten Cysteinmoleküle co-immobilisiert werden, um genügenden Abstand zwischen den Glykosiden sicherzustellen: auf der Monolage konnte führte eine Lösung mit 160µg/ml WGA auf der QCM nur zu einer Frequenzantwort von -30Hz, wogegen im Fall der Co-Immobilisierung -210 Hz, also das Siebenfache, erreicht werden konnten. Diese – für die vorliegende Studie maximale – Lösungskonzentration führt zu einer fast kompletten Monolage von WGA (0,98 Monolagen) auf der Sensoroberfläche. Daher lassen sich die Bindungskonstanten mittels eines Langmuir-Modells berechnen. Dazu wurde die Sensorcharakteristik in einem Konzentrationsbereich von 1 µg/ml bis 160 µg/ml aufgenommen. Im Gegensatz dazu adsorbieren MIP bereits bei niedrigen Lösungskonzentrationen (1 µg/ml) bereits Multilayer von WGA, womit ein modifiziertes BET-Modell für die Charakterisierung der Adsorptionsotherme herangezogen wurde. Das Adsorptionsverhalten legt den Schluß nahe, daß das MIP als „Kristallisationskeim“ für das Protein fungiert, obwohl das nichtgeprägte Material die Adsorption nicht begünstigt. Dies ist insbesondere auch daran zu bemerken, daß bis zu 25 Molekullagen auf dem MIP adsorbieren, wogegen die jeweiligen ungeprägten Referenzelektroden positive Frequenzantworten und damit Anti-Sauerbrey-Verhalten zeigen. Beim glykosidischen Rezeptoranalogon wird dagegen trotz maximal eine Monolage durch die Sensorschicht geboten, obwohl ein WGA-Molekül mehrere Bindungsstellen hat. Damit läßt sich dessen Verhalten durch eine Langmuir-Isotherme beschreiben. Im Vergleich der beiden Methoden lassen sich auf den MIP wesentlich höhere Sensoreffekt erzielen: beispielsweise bei 160µg/ml WGA sind es -1320 Hz im Vergleich zu -210 Hz für den Rezeptor. Die Selektivität gegenüber Bovinem Serumalbumin (BSA) ist dagegen mit eine Selektivitätsfaktor von drei im Fall des MIP niedriger, als für da Glykosid, das sieben erreicht. Daher ist bei diesem also die Selektivität besser, die Sensitivität dagegen geringer, als beim MIP.Wheat germ agglutinin (WGA) is a plant lectin that plays a crucial role in biotechnology and biosensors as it interacts with viruses, cells during infection events and also with oligosaccharides that are normally found on cell surfaces of several organisms. Therefore, it is an interesting analyte for mass sensitive sensing in terms of recognition and interaction phenomena. Within this work, WGA was selectively detected by two different techniques including immobilized carbohydrates and molecularly imprinted polymers (MIP) used as recognition elements on quartz crystal microbalance (QCM). Scanning tunneling microscopy (STM) was used to study the surfaces generated by those techniques. As STM requires a conductive surface, gold was sputtered onto the polymer for generating the STM image. Thus cavities having the dimension of WGA dimer could be observed in the resulting molecularly imprinted polymers (MIP). In the case of immobilized receptor analogues, N-acetyl-D-glucosamine (GlcNAc) modified with p-nitrophenol-cysteine was immobilizated on gold for construction of the sensitive layer. Due to the size of WGA molecules, a self-assembled monolayer of GlcNAc is unsuitable for binding as enough space is needed between binding sites to achieve optimal results. Therefore, cysteine molecules were used as spacers between each GlcNAc. Sensor characteristics reveal that the frequency decreased only by -30 Hz if pure GlcNAc is immobilized, which changed to roundly -210 Hz for the mixed surface layer at 160 µg/ml of WGA which in factor of 7. Furthermore, the maximum adsorption layer at the high concentration of WGA is nearly a monolayer (0.98 layers). Therefore, the binding constant was calculated with the Langmuir model. In parallel, WGA was used as the template for molecular imprinting in methacrylate co-polymer system. The sensor characteristics were recorded from 160 µg/ml to 1 µg/ml of WGA. In contrast to this, the adsorption behavior of WGA on the MIP surface occurred in multilayers starting from low concentrations (1 µg/ml). The studies on the WGA–MIP adsorption behavior suggest that the MIP itself works as a “crystallizing nucleus” for the protein, even though the nonimprinted material disfavors WGA adsorption. This can be seen by the fact that up to 25 molecular layers of protein are deposited on the MIP in the observed concentration range, whereas the NIP coated electrodes yield positive frequency shifts indicating anti-Sauerbrey behavior. In terms of adsorption investigation, the BET isotherm was applied to MIP for evaluating the binding properties of WGA due to this multilayer adsorption. Even though the interaction of WGA on glycoside surface is the multivalent interaction but the adsorption layer is occurred only monolayer adsorption leads to applying of Langmuir adsorption model. In comparison, the MIP method shows a substantially higher sensitivity (-1320 Hz for 160 µg/ml) than the immobilized receptor analogue (-210 Hz for 160 µg/ml). In terms of selectivity towards bovine serum albumin (BSA), the MIP has lower selectivity which reaches a factor of ~3 than the artificial receptor yields ~7. Therefore, this investigation indicated that the carbohydrate is better than the MIP in term of selectivity whereas MIP yields higher than artificial receptor for sensitivity

Adsorption isotherms and structure of cationic surfactants adsorbed on mineral oxide surfaces prepared by atomic layer deposition

The adsorption isotherms and aggregate structures of adsorbed surfactants on smooth thin-film surfaces of mineral oxides have been studied by optical reflectometry and atomic force microscopy (AFM). Films of the mineral oxides of titania, alumina, hafnia

Coadsorption of low molecular weight aromatic and aliphatic alcohols and acids with the cationic surfactant, CTAB, on silica surfaces

We have investigated the coadsorption of a range of small molecules with the cationic surfactant CTAB to silica surfaces over a range of concentrations and CTAB to solute ratios and compared the coadsorption with adsorption in the presence of the salicylate ion. We find that molecules with aromatic character and molecules with double bonds are most favorably adsorbed, and we attribute this to cation-π bonding between the surfactant headgroups and the π orbitals of the unsaturated bonds of the solute molecules. The adsorption is complex and depends on chemical interactions between the solute molecules and the surfactant, which are highly specific to the structure of the solute. To improve our understanding of the specifics of these interactions, we have performed one-dimensional rotating frame Overhauser spectroscopy (ROESY) nuclear magnetic resonance experiments. These experiments show the complexity of the intermolecular interactions and can be used to determine the position of the solute molecule with regard to the CTAB molecules in the adsorbed aggregates. The ROESY spectrum for the salicylate anion is distinct from those of the other solute molecules and suggests that the anions are dimerizing. Along with the cation-π bonding between the dimers, this provides a model for the strong influence that salicylate has on adsorption, micellar structure, and viscoelasticity. The ROESY data indicate that the catechol molecule interacts with all parts of the surfactant alkane chains such that they wrap around the molecule, but this has little effect on the interfacial curvature or aggregate shape. More intense isophthalic acid-CTAB intermolecular ROEs compared to those of other aromatic solutes are consistent with an interaction between isophthalic acid and the headgroups of two surfactant molecules that slows the intramicellar motion of isophthalic acid. Differences in interactions between solute molecules and the aliphatic surfactant chains do not result in changes in micelle structure

Adsorption isotherms and structure of cationic surfactants adsorbed on mineral oxide surfaces prepared by atomic layer deposition

The adsorption isotherms and aggregate structures of adsorbed surfactants on smooth thin-film surfaces of mineral oxides have been studied by optical reflectometry and atomic force microscopy (AFM). Films of the mineral oxides of titania, alumina, hafnia, and zirconia were produced by atomic layer deposition (ALD) with low roughness. We find that the surface strongly influences the admicelle organization on the surface. At high concentrations (2 x cmc) of cetyltrimethylammonium bromide (CTAB), the surfactant aggregates on a titania surface exhibit a flattened admicelle structure with an average repeat distance of 8.0 ± 1.0 nm whereas aggregates on alumina substrates exhibit a larger admicelle with an average separation distance of 10.5 ± 1.0 nm. A wormlike admicelle structure with an average separation distance of 7.0 ± 1.0 nm can be observed on zirconia substrates whereas a bilayered aggregate structure on hafnia substrates was observed. The change in the surface aggregate structure can be related to an increase in the critical packing parameter through a reduction in the effective headgroup area of the surfactant. The templating strength of the surfaces are found to be hafnia > alumina > zirconia > titania. Weakly templating surfaces are expected to have superior biocompatibility

Coadsorption of Low-Molecular Weight Aromatic and Aliphatic Alcohols and Acids with the Cationic Surfactant, CTAB, on Silica Surfaces

We have investigated the coadsorption of a range of small molecules with the cationic surfactant CTAB to silica surfaces over a range of concentrations and CTAB to solute ratios and compared the coadsorption with adsorption in the presence of the salicylate ion. We find that molecules with aromatic character and molecules with double bonds are most favorably adsorbed, and we attribute this to cation−π bonding between the surfactant headgroups and the π orbitals of the unsaturated bonds of the solute molecules. The adsorption is complex and depends on chemical interactions between the solute molecules and the surfactant, which are highly specific to the structure of the solute. To improve our understanding of the specifics of these interactions, we have performed one-dimensional rotating frame Overhauser spectroscopy (ROESY) nuclear magnetic resonance experiments. These experiments show the complexity of the intermolecular interactions and can be used to determine the position of the solute molecule with regard to the CTAB molecules in the adsorbed aggregates. The ROESY spectrum for the salicylate anion is distinct from those of the other solute molecules and suggests that the anions are dimerizing. Along with the cation−π bonding between the dimers, this provides a model for the strong influence that salicylate has on adsorption, micellar structure, and viscoelasticity. The ROESY data indicate that the catechol molecule interacts with all parts of the surfactant alkane chains such that they wrap around the molecule, but this has little effect on the interfacial curvature or aggregate shape. More intense isophthalic acid–CTAB intermolecular ROEs compared to those of other aromatic solutes are consistent with an interaction between isophthalic acid and the headgroups of two surfactant molecules that slows the intramicellar motion of isophthalic acid. Differences in interactions between solute molecules and the aliphatic surfactant chains do not result in changes in micelle structure