<p> </p><p>Control of protein immobilization at sensor surfaces is of great interest within various scientific fields, since it enables studies of specific biomolecular interactions. To achieve this, one must be able to immobilize proteins with retained native structure, while minimizing non-specific protein binding. The high affinity interaction between streptavidin (SA) and biotin is extensively used as a linker between a surface, where SA is immobilized, and the (biotinylated) molecule of interest. Self- assembled monolayers (SAMs) of poly- and oligo ethylene glycol (PEG and OEG) derivatives have been proven in literature to minimize non-specific protein binding, and biotin-exposing SAMs have been shown efficient for immobilization of SA.</p><p>The aim of this master's thesis project was to develop biotinylated gold surfaces for quartz crystal microbalance with dissipation monitoring (QCM-D) applications through the self-assembly of mixed monolayers of thiolated OEG (or PEG) derivatives with or without a terminal biotin head group. For this, different thiol compounds were to be compared and evaluated. For the systems under study, the required biotin density for maximum specific SA immobilization was to be established, while keeping the non-specific serum adsorption at a minimum. Model experiments with biotinylated proteins immobilized to the SA-functionalized surfaces were to be performed to evaluate the possibilities for commercialization.</p><p>A protocol for the preparation of a novel biotinylated surface was developed based on the immersion of gold substrates in an ethanolic incubation solution of dithiols with OEG chains (SS-OEG and SS-OEG-biotin, 99:1) and found to give reproducible results with respect to low non-specific protein binding and immobilization of a monolayer of SA. The modified surfaces allowed for subsequent immobilization of biotinylated bovine serum albumin (bBSA) and biotinylated plasminogen (bPLG). PLG was the subject of a challenging case study, using a combination of QCM-D and surface plasmon resonance (SPR), where the immobilized protein was subjected to low molecular weight ligands that were believed to induce conformational changes. The high control of the surface chemistry allowed for the interpretation of the increased dissipation shift upon ligand binding in terms of conformational changes.</p><p>An obstacle before commercialization of the described biotinylated surfaces is that they do not seem stable for storage > 7 days. The reasons for this have to be investigated further.</p>

This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.

Control of protein immobilization at sensor surfaces is of great interest within various scientific fields, since it enables studies of specific biomolecular interactions. To achieve this, one must be able to immobilize proteins with retained native structure, while minimizing non-specific protein binding. The high affinity interaction between streptavidin (SA) and biotin is extensively used as a linker between a surface, where SA is immobilized, and the (biotinylated) molecule of interest. Self- assembled monolayers (SAMs) of poly- and oligo ethylene glycol (PEG and OEG) derivatives have been proven in literature to minimize non-specific protein binding, and biotin-exposing SAMs have been shown efficient for immobilization of SA. The aim of this master's thesis project was to develop biotinylated gold surfaces for quartz crystal microbalance with dissipation monitoring (QCM-D) applications through the self-assembly of mixed monolayers of thiolated OEG (or PEG) derivatives with or without a terminal biotin head group. For this, different thiol compounds were to be compared and evaluated. For the systems under study, the required biotin density for maximum specific SA immobilization was to be established, while keeping the non-specific serum adsorption at a minimum. Model experiments with biotinylated proteins immobilized to the SA-functionalized surfaces were to be performed to evaluate the possibilities for commercialization. A protocol for the preparation of a novel biotinylated surface was developed based on the immersion of gold substrates in an ethanolic incubation solution of dithiols with OEG chains (SS-OEG and SS-OEG-biotin, 99:1) and found to give reproducible results with respect to low non-specific protein binding and immobilization of a monolayer of SA. The modified surfaces allowed for subsequent immobilization of biotinylated bovine serum albumin (bBSA) and biotinylated plasminogen (bPLG). PLG was the subject of a challenging case study, using a combination of QCM-D and surface plasmon resonance (SPR), where the immobilized protein was subjected to low molecular weight ligands that were believed to induce conformational changes. The high control of the surface chemistry allowed for the interpretation of the increased dissipation shift upon ligand binding in terms of conformational changes. An obstacle before commercialization of the described biotinylated surfaces is that they do not seem stable for storage &gt; 7 days. The reasons for this have to be investigated further.

Plasmonic circuitry is considered as a promising solution-effectivetechnology for miniaturizing and integrating the next generation ofoptical nano-devices. The realization of a practical plasmonic circuitry strongly depends on the complete understanding of the propagation properties of two key elements: surface plasmons and electrons. The critical part constituting the plasmonic circuitry is a waveguide which can sustain the two information-carriers simultaneously. Therefore, we present in this thesis the investigations on the propagation of surface plasmons and the co-propagation of surface plasmons and electrons in single crystalline metal nanowires. This thesis is therefore divided into two parts. In the first part, we investigate surface plasmons propagating in individual thick penta-twinned crystalline silver nanowires using dual-plane leakage radiation microscopy. The effective index and the losses of the mode are determined by measuring the wave vector content of the light emitted in the substrate. Surface plasmon mode is determined by numerical simulations and an analogy is drawn with molecular orbitals compound with similar symmetry. Leaky and bound modes selected by polarization inhomogeneity are demonstrated. We further investigate the effect of wire geometry (length, diameter) on the effective index and propagation losses. On the basis of the results obtained during the first part, we further investigate the effect of an electron flow on surface plasmon properties. We investigate to what extend surface plasmons and current-carrying electrons interfere in such a shared circuitry. By synchronously recording surface plasmons and electrical output characteristics of single crystalline silver and gold nanowires, we determine the limiting factors hindering the co-propagation of electrical current and surface plasmons in these nanoscale circuits. Analysis of wave vector distributions in Fourier images indicates that the effect of current flow on surface plasmons propagation is reflected by the morphological change during the electromigration process. We further investigate the possible crosstalk between co-propagating electrons and surface plasmons by applying alternating current bias

Endotoxin or Lipopolysaccharide (LPS) is the invariant structural component of gram negative bacterial outer membranes and is the chief causative factor of Sepsis or endotoxic shock. Sepsis is a syndrome that has very high mortality rates even in this age of excellent therapeutics and critical patient care. The treatment for sepsis till date remains nonspecific and supportive due to lack of effective anti-endotoxic drugs. Sepsis is initiated when the circulating bacteria shed LPS from their cell envelopes. Shed LPS aggregates are recognized by LPS binding proteins and receptors, which activate the host's immune system. Uncontrolled and excessive stimulation of the host's immune system precipitates endotoxic shock which in advanced cases involving multiple system organ failure inevitably lead to patient's death. Many strategies have been tested out to combat this deadly affliction. One of the attractive clinical modalities in sepsis treatment is the use of peptides as LPS sequestering anti-endotoxic drugs. A classical peptide antibiotic of this class is Polymyxin B (PMB) a cyclic cationic acylated molecule, that recognizes LPS with a very high affinity. This thesis describes kinetics and thermodynamics of PMB-LPS interactions and applies these parameters over a framework of different models so as to gain insights into the structure-function relationships that govern the interactions of this peptide with endotoxin(s). Classical biophysical techniques like fluorescence, circular dichroism spectroscopy, stopped flow kinetics, titration calorirnetry (ITC) and the relatively new technique of Surface Plasmon Resonance (SPR) have been employed to dissect out the mechanism of the range of non-covalent forces that are involved in peptide-endotoxin recognition. Certain proteins that exhibit LPS binding activity have also been studied to gains insight about their mode of action. Implications of these studies for designing peptides that have better anti-endotoxic properties are also highlighted. The first chapter introduces and highlights the clinical features of sepsis. It also attempts to shed light on the LPS mediated signal transduction pathway that leads to endotoxic shock. This chapter also briefly explains the roles of many LPS receptors that are present in the human system and their specific roles in the signal transduction pathways. The second part of this chapter deals with the role of cationic peptides as anti-endotoxic drugs. Certain key functional aspects of these peptides, which impart in them, the desirable property of LPS recognition have also been discussed The second chapter describes the kinetic studies undertaken to unravel the exact mechanism of LPS-PMB interaction. The studies reveal that PMB recognizes LPS in a biphasic manner, with the second, unimolecular isomerization step of the reaction being the rate-limiting step. The initial reaction is shown to be influenced by the presence of salt in the reaction medium. The dissociation phase of this interaction also shows a biphasic pattern. These data allow us to speculate upon the exact mechanism by which PMB is able to recognize LPS. The studies also shed light on some structural aspects that govern and confer such high LPS binding activity to PMB. Based on these a model has been proposed to explain this recognition (C.J. Thomas et al, 1998). The second chapter discuses the mode of action of various PMB analogs. These analogs have been chosen in terms of their mode of action as well as their structural similarly to PMB. The affinities of these analogs to LPS and lipid A were quantified using the Surface plasmon resonance (SPR) method. SPR, a technique that relies on the quantification of change in mass during a binary binding process occurring between an immobilized entity and a flowing ligand, is a rapid and sensitive method to measure biologically relevant interactions. SPR studies provide us with the binding constants and thermodynamic parameters that allow evaluation of the affinities of these peptides towards LPS (C.J.Thomas and A.Surolia, 1999). The third chapter discusses a hitherto unknown mode by which PMB acts on a LPS lamellae. The results of this study wherein the binding affinities of PMB and its analogs were performed on monolayers and tethered liposomes, show that PMB is able to remove specifically LPS or lipid A from monolayers or bilayer assemblies such as tethered liposomes. The exact mode of action of PMB is deciphered in the light of these new studies, which allow us to posit on the observed efficacy of PMB in neutralizing the endotoxin as compared to peptides with nearly similar affinities for LPS (C.J Thomas et al 1999). In the fourth chapter a series of 23 residue peptides, based on the sequence corresponding to the anti-sense strand of magainin gene have been synthesized. Magainin an amphiphilic helical peptide obtained from frog skins plays a vital role in the innate immune defense mechanisms of these organisms. It also exhibits LPS binding activity that makes it an attractive target as an anti-endotoxic drug. Biochemical and biophysical characterization of these peptides reveal that they have the tendency to perturb both the inner and the outer membranes of E.coli. The peptides are amphiphilic and have helical structure in a membrane bound environment. Three of the peptides tested have high affinities for lipid A that approach the values shown by PMB. The kinetic parameters obtained by stopped flow and SPR studies in conjunction with the therrnodynamic parameters obtained using ITC studies allow us to highlight the key structural features that need to be exhibited by peptides that are designed to be LPS recognizers. The studies also project the fact that ionic forces play an important role in the initial recognition of LPS by these peptides. Fortification of the might of these ionic charges increases affinity for LPS where as the hydrophobic residues that interact at the next phase of binding are more amenable to disruptions in contiguity. These factors are discussed using the helical wheel diagram that shows the clear amphiphilicity displayed by these peptides. (C.J Thomas et al Manuscript under preparation, 2000) Chapter six discusses the mode of action of certain LPS binding proteins. Limulus anti endotoxic factor (LALF) plays a vital role in the innate immune based defense systems of the horseshoe crab. Galectin-3 is a metal ion independent, galactosc binding Icctin of human origin with unknown functions. Both these phylogcntically-unrclatcd proteins exhibit LPS/lipid A recognizing properties. ITC and SPR studies have been used to determine the binding constants displayed by these proteins for lipid A. LALF bind to lipid A with very high affinity than compared to Galectin-3 and is also able to take away selectively lipid A from both monolayers and tethered liposomes. Galectin-3 does not show this property of LALF, which might account for its lowered affinities. Also structurally LALF has amphiphilic nature that confers high lipid A binding activity, which is clearly lacking in Galectin-3. These studies in conjunction with the knowledge gained from the study of LPS-PMB interaction stress on the importance of amphiphilicity in LPS recognition. (C.J Thomas et al Manuscript under preparation, 2000). The final chapter is a general discussion that attempts to collate all these kinetic and thermodynamic observations in the pursuit of designing small easily manipulatable peptides that exhibit high LPS binding activity. These studies are aimed to act as rough guidelines to the design of LPS sequestering peptides that might have better therapeutic and pharmacokinetic properties. The appendix to the main body of work presented in thesis are two pieces of work pertaining to the elucidation the kinetics and mechanism of sugar lectin interactions, when sugars are presented as glycolipids in monolayers or bilaycrs liposomes. Mode of the presentation of sugars at cell-surfaces in the form of glycolipids as ligands influence their recognition by macromolecular receptors like lectins. Appendix 1 is a study of the mode of action of Ulex europeus I lectin binding to H-fucolipid containing tethered liposomes, by SPR. Fucosylated sugars are often used as key markers in histochemical analysis of malignant cancerous tissues. Ulex lectin plays a vital role as a marker for identification of these tissues. The kinetics and thermodynamic parameters that are obtained in this study throw some light on the mode of recognition of glycolipid receptor by Ulex europeus I lectin (C.J Thomas and A. Surolia 2000). Appendix 2 is a study, that attempts to quantify the initial kinetic parameters that correlate the recognition of glycolipid receptors with their inclination at the membrane surface and the influence of charge on them by soyabean agglutinin (SBA), Abrus agglutinin I and II. Studies on the soyabean agglutinin-globoside interaction highlights the divalent cation mediated reorientation of these receptors on their accessibility and recognition to the agglutinin. The divalent cations are speculated to orient the oligosaccharide head groups in a spatial geometry that allows a heightened kinetics of their interaction by SBA. These studies reveal that the reorganization of the binding pocket of a lectin can also have a profound influence on ihc rates of recognition of a glycospingolipid ligand by a lectin as exemplified by Abrus agglutinin II- GM1 interactions (C.J Thomas ct al, Manuscript under preparation).

Im Mittelpunkt dieser Arbeit stand die Darstellung sensitiver Blockcopolymere und deren Gele, die als Ausgangsmaterialien in Sensor- und Aktorsystemen einsetzbar sind. Die Vereinigung verschiedener Ansprechparameter stellt erhöhte Anforderung an die Synthese. Geringe Ansprechzeiten lassen sich mit einer Gelgröße im µm-Bereich erreichen. Hydrogele dieser Größenordnungen können durch nachträgliche Vernetzung funktioneller linearer Polymere ermöglicht werden. Die Makroinitiatormethode ermöglichte den Aufbau verschiedener linearer photovernetzbarer Blockcopolymere. Zum Einen wurde das temperatursensitive P(n-BuAc)-block-P(PNIPAAm-co-DMIAAm) erhalten, des Weiteren gelang die Darstellung der multi-sensitiven Blockcopolymere P2VP-block-P(NIPAAm-co-DMIAAm) und P4VP-block-P(NIPAAm-co-DMIAAm). Die Blockcopolymere wurden mit variierenden Blocklängen und Verhältnissen sowie mit unterschiedlichem Vernetzergehalt dargestellt. Die Charakterisierung der Blockcopolymere erfolgte mittels 1H-NMR-Spektroskopie, GPC-Messungen (Zusammensetzung) und DSC-Messungen (thermische Eigenschaften). Das Löslichkeitsverhalten in wässrigen Medien wurde durch Dynamische Lichtstreuung bestimmt. Die Beschreibung des Quellverhaltens der vernetzten Schichten erfolgte durch vornehmlich durch optische Methoden (SPR/OWS, WAMS, Ellipsometrie). Die Veränderung des E-Moduls in Abhängigkeit äußerer Parameter konnte mittels AFM untersucht werden. Die Reaktion der Schichten wurde gegenüber Temperatur, pH-Wert und Salzkonzentrationen getestet. Die charakterisierten Filme konnten im Anschluss als sensitive Schichten in piezoresistiven Sensorsystemen verwendetet werden.

Metal nanoparticles have recently gained popularity in many research areas due to their nanosize-related properties. Depending on the size of the metal nanoparticle, their mode of interaction with electromagnetic radiation and the outcome of this interaction vary; in turn the effect exerted on a protein which is conjugated to a nanoparticle varies, because different sized nanoparticles demonstrate different modes of energy transfer with electromagnetic radiation and molecules conjugated to them. Very small cluster with sizes around 1 – 1.2 nm tend to get excited by incident light and emit fluorescence, whereas larger nanoparticles absorb the incoming light very strongly due to their LSPR. In this study we observed the outcomes of the interaction between two types of nanoparticles, namely gold and gold/silver alloyed nanoparticles with the fluorescence emission of two fluorophores, namely eGFP and rPhiYFP; and demonstrated a bioassay where the fluorescence modulation by gold nanoparticles can be used as the sensing strategy. Lastly, we demonstrated the potential of autofluorescent gold nanoparticles as intracellular reporters.

Surface polaritons, i.e., collective oscillations of the surface charges, strongly influence the optical response at the micro- and nanoscale and have to be accounted for in modern nanotechnology. Within this thesis, certain basic phenomena involving surface polaritons are investigated by means of the semianalytical multiple-multipole (MMP) method. The results are compared to experiments. In the first part, the surface plasmon resonance (SPR) of metal nanoparticles is analyzed. This resonant collective oscillation of the free electrons in a metallic nanoparticle leads to an enhancement and confinement of the local electric field at optical frequencies. The local electric field can be further increased by tailoring the shape of the particle or by using near-field-interacting dimers or trimers of gold nanospheres. The hot spots found under such conditions increase the sensitivity of surface-enhanced Raman scattering by several orders of magnitude and simultaneously reduce the probed volume, thereby providing single-molecule sensitivity. The sub-wavelength-confined strong electromagnetic field associated with a SPR provides the basis for scattering-type near-field optical microscopy or tip-enhanced Raman spectroscopy, where the metal particle serves as a probe that is scanned laterally in the vicinity of a substrate. The presence of the latter causes a characteristic shift of the SPR towards lower frequencies. This effect originates in the near-field interaction of the surface charges on the objects. Furthermore, the excitation of higher-order modes becomes possible in case of an excitation by a strongly inhomogeneous wave, such as an evanescent wave. These modes may significantly contribute to the near field but have only very little influence on the far-field signature. Instead of using resonant probes, one may place a nonresonant probe in the vicinity of a substrate having a high density of electromagnetic surface states. This also produces a resonance of the light scattering by the system. Especially polar crystals, such as the investigated silicon carbide, feature such a high density of surface phonon polariton states in the mid-infrared spectral region, which can be excited due to the near-field interaction with a polarized particle. Thereby, a resonance is created leading to a strong increase of the electric field at the interface. In the second part of the thesis, special emphasis is put on surface plasmon polaritons (SPPs). Such propagating surface waves can be excited directly by plane waves only at patterned interfaces. This process is studied for the case of a groove. The groove breaks the translational invariance, so that the SPPs can be launched locally at the edges of the groove. Additionally, the mode(s) inside the groove are excited. These modes can basically be understood as metal-insulator-metal cavity modes. Their dispersion strongly depends on the groove width. The cavity behavior caused by the finite depth provides another degree of freedom for optimizing the SPP excitation by plane waves. Thin metallic films deposited on glass offer two different SPP waveguide modes, each of which can be addressed preferentially by a proper choice of the width of the groove. The reflection, transmission, scattering, and the conversion of the modes at discontinuities such as edges, steps, barriers, and grooves can be controlled by appropriately designing the geometry at the nanoscale. Furthermore, the excitation of SPPs at single and multiple slits in thin-film metal waveguides on glass and their propagation and scattering is shown by scanning near-field optical experiments. Such waveguide structures offer a means for transporting light in a confined way. Especially triangularly shaped waveguides can be used to guide light in sub-wavelength spaces. / Die Wechselwirkung von elektromagnetischer Strahlung mit subwellenlängenkleinen Teilchen bzw. Oberflächenstrukturen ermöglicht nicht nur eine Miniaturisierung optischer Geräte, sondern erlaubt sehr interessante Anwendungen, beispielsweise in der Sensorik und Nahfeldoptik. In der vorliegenden Arbeit werden die zu Grunde liegenden Effekte im Rahmen der klassischen Elektrodynamik mit Hilfe der semianalytischen Methode der multiplen Multipole (MMP) analysiert, und die Ergebnisse werden mit Experimenten verglichen. Im ersten Teil werden Oberflächenplasmonenresonanzen (engl. surface plasmon resonance - SPR) einzelner und wechselwirkender Metallteilchen untersucht. Die dabei auftretende resonante kollektive Schwingung der freien Elektronen des Partikels bewirkt eine deutliche Erhöhung und Lokalisierung des elektromagnetischen Feldes in seiner Umgebung. Die spektrale Position und die Stärke der SPR eines Nanoteilchens, die von dessen geometrischer Form, Permittivität und Umgebung abhängen, können nur im Grenzfall sehr kleiner Teilchen elektrostatisch beschrieben werden, wohingegen der verwendete semianalytische MMP-Ansatz weitaus flexibler ist und insbesondere auch auf größere Partikel, Teilchen mit komplizierterer Form bzw. Ensembles von Partikeln anwendbar ist. Die betrachteten einzelnen kleinen (&amp;lt; Wellenlänge) Goldkügelchen und Silberellipsoide besitzen eine stark ausgeprägte SPR im sichtbaren optischen Bereich. Diese ist auf eine dipolartige Polarisierung des Teilchens zurückzuführen. Höhere Moden der Polarisation können entweder als Folge von Retardierungseffekten an größeren (mit der Wellenlänge vergleichbaren) Teilchen oder bei der Verwendung inhomogener (z.B. evaneszenter) Wellen angeregt werden. Partikel, die sich in der Nähe eines Substrates befinden, unterliegen der Nahfeldwechselwirkung zwischen den (lichtinduzierten) Oberflächenladungen auf der Oberfläche des Teilchens und des Substrats. Dies führt zu einer Verschiebung der SPR zu niedrigeren Frequenzen und einer Erhöhung des lokalen elektrischen Feldes. Letzteres bildet die Grundlage z.B. der spitzenverstärkten Raman-Spektroskopie und der optischen Nahfeldmikroskopie mit Streulichtdetektion. Dasselbe Prinzip bewirkt ein stark überhöhtes elektrisches Feld zwischen miteinander wechselwirkenden Nanopartikeln, welches z.B. die Sensitivität der oberflächenverstärkten Raman-Mikroskopie um mehrere Größenordnungen steigern kann. Im Gegensatz zur SPR einzelner Nanopartikel kann die Resonanz der Lichtstreuung im Fall eines Partikels in der Nähe eines Substrats aus der durch die Nahfeldwechselwirkung induzierten Anregung elektromagnetischer Oberflächenzustände entstehen. Diese wirken ihrerseits auf das Nanopartikel zurück, wobei eine resonante Lichtstreuung beobachtbar ist. Dieser, am Beispiel einer metallischen Nahfeldsonde über einem Siliziumcarbid-Substrat analysierte, Effekt ermöglicht bei einer ganzen Klasse von polaren Kristallen interessante Anwendungen in der Mikroskopie und Sensorik basierend auf der hohen Dichte von Oberflächenphononpolaritonen dieser Kristalle im mittleren infraroten Spektralbereich und deren nahfeldinduzierten Anregung. Im zweiten Teil der Arbeit werden kollektive Anregungen von Elektronen an Metalloberflächen untersucht. Die dabei auftretenden plasmonischen Oberflächenwellen (engl. surface plasmon polaritons - SPPs) weisen einen exponentiellen Abfall der Intensität senkrecht zur Grenzfläche auf. Diese starke Lokalisierung der Energie an der Oberfläche bildet die Grundlage vieler Anwendungen, z.B. im Bereich der hochempfindlichen Detektion (bio)chemischer Verbindungen oder für eine zweidimensionale Optik (engl. plasmonics). Das Aufheben der Translationsinvarianz längs der Oberfläche ermöglicht die direkte Anregung von SPPs durch ebene Wellen. Die Abhängigkeit dieser Kopplung von der Geometrie wird am Beispiel eines Nanograbens untersucht. Dabei werden neben den SPPs ebenfalls eine oder mehrere Moden im Graben angeregt. Folglich ermöglicht die geeignete Wahl der Grabengeometrie die Optimierung der Umwandlung von ebenen Wellen in SPPs. Im - in der Praxis weit verbreiteten - Fall asymmetrisch eingebetteter metallischer Dünnschichtwellenleiter existieren zwei Moden. In Abhängigkeit von der Grabenbreite kann die eine oder die andere Mode bevorzugt angeregt werden. Die Analyse der Wechselwirkung von SPPs mit Oberflächenstrukturen, z.B. Kanten, Stufen, Barrieren und Gräben, zeigt die Möglichkeit der Steuerung der Reflexions-, Transmissions- und Abstrahleigenschaften durch die gezielte Wahl der Geometrie der &amp;quot;Oberflächendefekte&amp;quot; auf der Nanoskala und deckt die zu Grunde liegenden Mechanismen und die daraus resultierenden Anforderungen bei der Herstellung neuer plasmonischer Komponenten auf. Exemplarisch wird das Prinzip der SPP-Anregung an einzelnen und mehreren Gräben in dünnen metallischen Filmen sowie der subwellenlängen Feldlokalisierung an sich verjüngenden metallischen Dünnschichtwellenleitern unter Verwendung der optischen Nahfeldmikroskopie experimentell gezeigt.

It is widely known that early estimates about the binding properties of drug candidates are important in the drug discovery process. Surface plasmon resonance (SPR) biosensors have become a standard tool for characterizing interactions between a great variety of biomolecules and it offers a unique opportunity to study binding activity. The aim of this project was to develop a SPR based assay for pre-screening of low molecular weight (LMW) drug compounds, to enable filtering away disturbing compounds when interacting with drugs. The interaction between 47 LMW compounds and biological ligands were investigated using the instrument BiacoreTM, which is based on SPR-technology.

Les microcantileviers fonctionnalisés offrent une plateforme idéale pour la nano- et micro-mécanique et pour le développement de (bio-) capteurs tres sensible. Le principe d’opération consiste dans des évènements physicochimiques qui se passent du côté fonctionnalisé du microcantilevier induisant une différence de stress de surface entre les deux côtés du cantilevier qui cause une déflexion verticale du levier. Par contre, les facteurs et les phénomènes interfacials qui régissent la nature et l'intensité du stress de surface sont encore méconnus. Pour éclaircir ce phénomène, la première partie de cette thèse porte sur l'étude des réactions de microcantileviers qui sont recouverts d'or et fonctionnalisés par une monocouche auto-assemblée (MAA) électroactive. La formation d'une MAA de ferrocènylundécanethiol (FcC11SH) à la surface d'or d'un microcantilevier est le modèle utilisé pour mieux comprendre le stress de surface induit par l’électrochimie. Les résultats obtenus démontrent qu'une transformation rédox de la MAA de FcC11SH crée un stress de surface qui résulte dans une déflexion verticale du microcantilevier. Dépendamment de la flexibilité du microcantilevier, cette déflexion peut varier de quelques nanomètres à quelques micromètres. L’oxydation de cette MAA de FcC11SH dans un environnement d'ions perchlorate génère un changement de stress de surface compressive. Les résultats indiquent que la déflexion du microcantilevier est due à une tension latérale provenant d'une réorientation et d'une expansion moléculaire lors du transfért de charge et de pairage d’anions. Pour vérifier cette hypothèse, les mêmes expériences ont été répéteés avec des microcantileviers qui ont été couverts d'une MAA mixte, où les groupements électroactifs de ferrocène sont isolés par des alkylthiols inactifs. Lorsqu’un potentiel est appliqué, un courant est détecté mais le microcantilevier ne signale aucune déflexion. Ces résultats confirment que la déflexion du microcantilevier est due à une pression latérale provenant du ferrocènium qui se réorganise et qui crée une pression sur ses pairs avoisinants plutôt que du couplage d’anions. L’amplitude de la déflexion verticale du microcantilevier dépend de la structure moléculaire de la MAA et du le type d’anion utilisés lors de la réaction électrochimique. Dans la prochaine partie de la thèse, l’électrochimie et la spectroscopie de résonance de plasmon en surface ont été combinées pour arriver à une description de l’adsorption et de l’agrégation des n-alkyl sulfates à l’interface FcC11SAu/électrolyte. À toutes les concentrations de solution, les molécules d'agent tensio-actif sont empilées perpendiculairement à la surface d'électrode sous forme de monocouche condensé entrecroisé. Cependant, la densité du film spécifiquement adsorbé s'est avérée être affectée par l'état d'organisation des agents tensio-actifs en solution. À faible concentration, où les molécules d'agent tensio-actif sont présentes en tant que monomères solvatés, les monomères peuvent facilement s'adapter à l’évolution de la concentration en surface du ferrocènium lors du balayage du potential. Cependant, lorsque les molécules sont présentes en solution en tant que micelles une densité plus faible d'agent tensio-actif a été trouvée en raison de l'incapacité de répondre effectivement à la surface de ferrocenium générée dynamiquement. / Surface-functionalized microcantilevers provide an ideal platform for nano- and micro-mechanical actuation and highly sensitive sensing technologies. The basic principle of operation is that a chemical or physical event occurring at the functionalized surface of one side of the cantilever generates a surface stress difference (between the active functionalized and passive non-functionalized sides) that causes the cantilever to bend away from its resting position. However, the factors and phenomena contributing to both the nature and magnitude of the surface stress are not well understood. To this end, the first part of this thesis focused on investigating the potential-controlled actuation and surface stress properties of free-standing gold-coated microcantilevers functionalized with a redox-active self-assembled monolayer (SAM). A ferrocenylundecanethiolate (FcC11SAu) SAM on a gold-coated cantilever was used as a model system to investigate the surface stress generated by faradaic chemistry. The data obtained clearly demonstrates that the electrochemical transformation of a ferrocene moiety in a monomolecular organic film can generate a surface stress change of sufficient magnitude to deflect a microcantilever. In fact, depending on the flexibility of the microcantilever, the mechanical deflection resulting from the redox transformation of the surface-tethered ferrocene can range on the order of nanometers to micrometers. The oxidation of the FcC11SAu SAM in perchlorate electrolyte generates a compressive surface stress change. The microcantilever deflection is driven by the lateral tension resulting from molecular reorientation/volume expansion accompanying the charge-transfer and ion-pairing events. To verify this hypothesis, mixed SAM-modified microcantilevers, in which the electroactive ferrocenes are isolated from one another by an inert n-alkylthiolate matrix, were investigated. Under an applied potential, a Faradaic current was measured, but no microcantilever beam deflection was observed. This finding confirms that the cantilever responds to the lateral pressure exerted by an ensemble of re-orienting ferrocenium-bearing alkylthiolates upon each other rather than to individual anion pairing events. Changes in molecular structure and anion type can also be used to modulate the extent of micromechanical motion. In the next part of the dissertation, electrochemical measurements and surface plasmon resonance spectroscopy were combined to present a description of the adsorption and aggregation of n-alkyl sulfates at the FcC11SAu/electrolyte interface. At all bulk solution concentrations, the surfactant moieties packed perpendicular to the electrode surface in the form of an interdigitated condensed film. However, the density of the specifically adsorbed film was found to be affected by the organizational state of the surfactants in solution. At low concentrations, where the surfactant molecules are present as solvated monomers, the monomers can readily adapt to the changing ferrocenium concentration with the potential potential scan. However, when the molecules are present as micellar structures in solution, a lower surfactant packing density was found because of the inability to respond effectively to the dynamically generated surface ferroceniums. This research demonstrates the potential utility of charge-transfer interactions for organizing materials at solid interfaces and effecting micromechanical actuation using an electrifical stimulus.