Systematic studies of protein immobilization by surface plasmon field-enhanced fluorescence spectroscopy

The research interest of this study is to investigate surface immobilization strategies for proteins and other biomolecules by the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) technique. The recrystallization features of the S-layer proteins and the possibility of combining the S-layer lattice arrays with other functional molecules make this protein a prime candidate for supramolecular architectures. The recrystallization behavior on gold or on the secondary cell wall polymer (SCWP) was recorded by SPR. The optical thicknesses and surface densities for different protein layers were calculated. In DNA hybridization tests performed in order to discriminate different mismatches, recombinant S-layer-streptavidin fusion protein matrices showed their potential for new microarrays. Moreover, SCWPs coated gold chips, covered with a controlled and oriented assembly of S-layer fusion proteins, represent an even more sensitive fluorescence testing platform. Additionally, S-layer fusion proteins as the matrix for LHCII immobilization strongly demonstrate superiority over routine approaches, proving the possibility of utilizing them as a new strategy for biomolecular coupling. In the study of the SPFS hCG immunoassay, the biophysical and immunological characteristics of this glycoprotein hormone were presented first. After the investigation of the effect of the biotin thiol dilution on the coupling efficiently, the interfacial binding model including the appropriate binary SAM structure and the versatile streptavidin-biotin interaction was chosen as the basic supramolecular architecture for the fabrication of a SPFS-based immunoassay. Next, the affinity characteristics between different antibodies and hCG were measured via an equilibrium binding analysis, which is the first example for the titration of such a high affinity interaction by SPFS. The results agree very well with the constants derived from the literature. Finally, a sandwich assay and a competitive assay were selected as templates for SPFS-based hCG detection, and an excellent LOD of 0.15 mIU/ml was attained via the “one step” sandwich method. Such high sensitivity not only fulfills clinical requirements, but is also better than most other biosensors. Fully understanding how LHCII complexes transfer the sunlight energy directionally and efficiently to the reaction center is potentially useful for constructing biomimetic devices as solar cells. After the introduction of the structural and the spectroscopic features of LHCII, different surface immobilization strategies of LHCII were summarized next. Among them the strategy based on the His-tag and the immobilized metal (ion) affinity chromatography (IMAC) technique were of great interest and resulted in different kinds of home-fabricated His-tag chelating chips. Their substantial protein coupling capacity, maintenance of high biological activity and a remarkably repeatable binding ability on the same chip after regeneration was demonstrated. Moreover, different parameters related to the stability of surface coupled reconstituted complexes, including sucrose, detergent, lipid, oligomerization, temperature and circulation rate, were evaluated in order to standardize the most effective immobilization conditions. In addition, partial lipid bilayers obtained from LHCII contained proteo-liposomes fusion on the surface were observed by the QCM technique. Finally, the inter-complex energy transfer between neighboring LHCIIs on a gold protected silver surface by excitation with a blue laser (λ = 473nm) was recorded for the first time, and the factors influencing the energy transfer efficiency were evaluated.

Thin functional conducting polymer films

In the present study, thin functional conducting polyaniline (PANI) films, either doped or undoped, patterned or unpatterned, were prepared by different approaches. The properties of the obtained PANI films were investigated in detail by a combination of electrochemistry with several other techniques, such as SPR, QCM, SPFS, diffraction, etc. The sensing applications (especially biosensing applications) of the prepared PANI films were explored. Firstly, the pure PANI films were prepared by the electropolymerisation method and their doping/dedoping properties in acidic conditions were investigated in detail by a combination of electrochemistry with SPR and QCM. Dielectric constants of PANI at different oxidation states were obtained quantitatively. The results obtained here laid a good foundation for the following investigations of PANI films in neutral pH conditions. Next, PANI multilayer films doped by a variety of materials were prepared by the layer-by-layer method in order to explore their biosensing applications, because of the loss of redox activity of pure PANI in neutral pH conditions. The dopants used include not only the traditionally used linear polyelectrolytes, but also, for the first tim, some other novel materials, like modified gold nanoparticles or modified carbon nanotubes. Our results showed that all the used dopants could form stable multilayer films with PANI. All the obtained PANI multilayer films showed good redox activity in a neutral pH environment, which makes them feasible for bioassays. We found that all the prepared PANI multilayer films can electrocatalyze the oxidation of NADH in neutral conditions at a low potential, although their catalytic efficiencies are different. Among them, PANI/carbon nanotube system showed the highest catalytic efficiency toward the oxidation of NADH, which makes it a good candidate as a NADH sensor. Besides, because some of the prepared PANI multilayer systems were end-terminated with –COOH groups (like PANI/Au nanoparticles system), which can be utilized to easily link biomolecules for biosensing applications. Here, we demonstrated, for the first time, to use the prepared PANI multilayer films for the DNA hybridisation detection. The detection event was monitored either by direct electrochemical method, or by enzyme-amplified electrochemical method, or by surface plasmon enhanced fluorescence spectroscopic method. All the methods can effectively differentiate non-complementary DNA from the complementary ones, even at the single-base mismatch level. It should also be noted that, our success in fabricating PANI multilayer films with modified Au nanoparticles or carbon nanotubes also offered another novel method for incorporating such novel materials into (conducting) polymers. Because of the unique electrochemical and optical properties of each component of the obtained PANI multilayer films, they should also find potential applications in many other fields such as microelectronics, or for electrochromic and photovoltaic devices. Finally, patterned PANI films were fabricated by the combination of several patterning techniques, such as the combination of electrocopolymerization with micromolding in capillaries (EP-MIMIC), the combination of microcontact printing with the layer-by-layer technique (µCP-LBL), and the polystyrene (PS) template induced electropolymerisation method. Using the obtained stripe-shaped PANI/PSS film, a redox-switchable polymer grating based on the surface-plasmon-enhanced mode was constructed and its application in the field of biosensing was explored. It was found that the diffraction efficiency (DE) of the grating was very sensitive to the applied potential (i.e. redox state of the film) as well as the pH environment of the dielectric medium. Moreover, the DE could also be effectively tuned by an electrocatalytic event, such as the electrocatalytic oxidation of NADH by the grating film. By using PS colloidal crystal assemblies as templates, well-ordered 3D interconnected macroporous PANI arrays (PANI inverse opals) were fabricated via electropolymerisation method. The quality of the obtained inverse opals was much higher than those reported by chemical synthesis method. By electrochemical method, the structures of the prepared inverse opals can be easily controlled. To explore the possible biosensing applications of PANI inverse opals, efforts were also done toward the fabrication of PANI composite inverse opals. By selecting proper dopants, high quality inverse opals of PANI composites were fabricated for the first time. And the obtained opaline films remained redox-active in neutral pH conditions, pointing to their possible applications for electrobioassays.

A hybrid technology for parallel recording of single ion channels

Hybrid technologies, thanks to the convergence of integrated microelectronic devices and new class of microfluidic structures could open new perspectives to the way how nanoscale events are discovered, monitored and controlled. The key point of this thesis is to evaluate the impact of such an approach into applications of ion-channel High Throughput Screening (HTS)platforms. This approach offers promising opportunities for the development of new classes of sensitive, reliable and cheap sensors. There are numerous advantages of embedding microelectronic readout structures strictly coupled to sensing elements. On the one hand the signal-to-noise-ratio is increased as a result of scaling. On the other, the readout miniaturization allows organization of sensors into arrays, increasing the capability of the platform in terms of number of acquired data, as required in the HTS approach, to improve sensing accuracy and reliabiity. However, accurate interface design is required to establish efficient communication between ionic-based and electronic-based signals. The work made in this thesis will show a first example of a complete parallel readout system with single ion channel resolution, using a compact and scalable hybrid architecture suitable to be interfaced to large array of sensors, ensuring simultaneous signal recording and smart control of the signal-to-noise ratio and bandwidth trade off. More specifically, an array of microfluidic polymer structures, hosting artificial lipid bilayers blocks where single ion channel pores are embededed, is coupled with an array of ultra-low noise current amplifiers for signal amplification and data processing. As demonstrating working example, the platform was used to acquire ultra small currents derived by single non-covalent molecular binding between alpha-hemolysin pores and beta-cyclodextrin molecules in artificial lipid membranes.

Depicting the role of CDKN2A/ARF alterations in adult BCR-ABL1-positive acute lymphoblastic leukemia patients: from genomic deletions to prognostic impact

This 9p21 locus, encode for important proteins involved in cell cycle regulation and apoptosis containing the p16/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15/CDKN2B. This locus, is a major target of inactivation in the pathogenesis of a number of human tumors, both solid and haematologic, and is a frequent site of loss or deletion also in acute lymphoblastic leukemia (ALL) ranging from 18% to 45% 1. In order to explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1-positive ALL and to investigate their prognostic value, 112 patients (101 de novo and 11 relapse cases) were analyzed by genome-wide single nucleotide polymorphisms arrays and gene candidate deep exon sequencing. Paired diagnosis-relapse samples were further available and analyzed for 19 (19%) cases. CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases and in 43% the minimal overlapping region of the lost area spanned only the CDKN2A/2B gene locus. The analysis at the time of relapse showed an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06). Point mutations within the 9p21 locus were found at very low level with only a non-synonymous substition in the exon 2 of CDKN2A. Finally, correlation with clinical outcome showed that deletions of CDKN2A/B are significantly associated with poor outcome in terms of overall survival (p = 0.0206), disease free-survival (p = 0.0010) and cumulative incidence of relapse (p = 0.0014). The inactivation of 9p21 locus by genomic deletions is a frequent event in BCR-ABL1-positive ALL. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker.

Structural and functional analysis of centromeric chromatin

Animal neocentromeres are defined as ectopic centromeres that have formed in non-centromeric locations and avoid some of the features, like the DNA satellite sequence, that normally characterize canonical centromeres. Despite this, they are stable functional centromeres inherited through generations. The only existence of neocentromeres provide convincing evidence that centromere specification is determined by epigenetic rather than sequence-specific mechanisms. For all this reasons, we used them as simplified models to investigate the molecular mechanisms that underlay the formation and the maintenance of functional centromeres. We collected human cell lines carrying neocentromeres in different positions. To investigate the region involved in the process at the DNA sequence level we applied a recent technology that integrates Chromatin Immuno-Precipitation and DNA microarrays (ChIP-on-chip) using rabbit polyclonal antibodies directed against CENP-A or CENP-C human centromeric proteins. These DNA binding-proteins are required for kinetochore function and are exclusively targeted to functional centromeres. Thus, the immunoprecipitation of DNA bound by these proteins allows the isolation of centromeric sequences, including those of the neocentromeres. Neocentromeres arise even in protein-coding genes region. We further analyzed if the increased scaffold attachment sites and the corresponding tighter chromatin of the region involved in the neocentromerization process still were permissive or not to transcription of within encoded genes. Centromere repositioning is a phenomenon in which a neocentromere arisen without altering the gene order, followed by the inactivation of the canonical centromere, becomes fixed in population. It is a process of chromosome rearrangement fundamental in evolution, at the bases of speciation. The repeat-free region where the neocentromere initially forms, progressively acquires extended arrays of satellite tandem repeats that may contribute to its functional stability. In this view our attention focalized to the repositioned horse ECA11 centromere. ChIP-on-chip analysis was used to define the region involved and SNPs studies, mapping within the region involved into neocentromerization, were carried on. We have been able to describe the structural polymorphism of the chromosome 11 centromeric domain of Caballus population. That polymorphism was seen even between homologues chromosome of the same cells. That discovery was the first described ever. Genomic plasticity had a fundamental role in evolution. Centromeres are not static packaged region of genomes. The key question that fascinates biologists is to understand how that centromere plasticity could be combined to the stability and maintenance of centromeric function. Starting from the epigenetic point of view that underlies centromere formation, we decided to analyze the RNA content of centromeric chromatin. RNA, as well as secondary chemically modifications that involve both histones and DNA, represents a good candidate to guide somehow the centromere formation and maintenance. Many observations suggest that transcription of centromeric DNA or of other non-coding RNAs could affect centromere formation. To date has been no thorough investigation addressing the identity of the chromatin-associated RNAs (CARs) on a global scale. This prompted us to develop techniques to identify CARs in a genome-wide approach using high-throughput genomic platforms. The future goal of this study will be to focalize the attention on what strictly happens specifically inside centromere chromatin.

Influence of genetic variants and post transcriptional factors on imatinib transporters as determinants of the pharmacological response

Introduction – Although imatinib (IM) is a recognized gold standard in chronic myeloid leukemia (CML) therapy, resistance has emerged in a significant proportion of patients. Aim – The aim of this study was: (1) to investigate the role of genetic variants in genes encoding for IM transporters, as candidate of IM responsiveness and (2) to test the influence of miRNAs on IM response, focusing on efflux transporters. Methods – As a first step, a panel of polymorphisms (SNPs) was genotyped in a subgroup population of 189 patients enrolled in the Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial. The association with cytogenetic response and molecular response (MR) was assessed for each SNP. As a second step, an in vitro IM-resistant model (K-562 CML cell line) was established. miRNAs profiles were analyzed using Taqman arrays and in silico search was performed for miRNAs deregulated after IM treatment. mRNA and protein expression were quantified using TaqMan realtime PCR and Western blotting, respectively. Results – (1) Among Caucasian patients, ABCB1 rs60023214 significantly correlated with complete MR (P = 0.005). Concerning SNPs combination in IM uptake transporters, the associations with treatment outcomes were statistically significant for both major and complete MR (P = 0.005 and P = 0.01, respectively). (2) ABCB1 protein was not expressed under any conditions of treatment, differently from ABCG2. Two deregulated miRNAs, namely miR-212 and miR-328, were identified to be inversely correlated with ABCG2 (r2= 0.57; p=0.03 and r2=0.47; p=0.06, respectively). Experiments of loss and gain of function confirmed the functional influence of these miRNAs on ABCG2. Conclusion – The multiple candidate gene approach identified single and combination of SNPs that can be proposed as predictor of IM response. The in vitro study suggested that IM resistance could be mediated by miRNA-dependent mechanism. Further studies are needed to validate these preliminary findings.

Silicon-containing block copolymers with well-defined, branched architectures

In the past decade, block copolymers (BCPs) have attracted increasing scientific and technological interest because of their inherent capability to spontaneously self-assemble into ordered arrays of nanostructures. The importance of nanostructures in a number of applications has fostered the need for well-defined, complex macromolecular architectures. In this thesis, the influence of macromolecular architecture on the bulk morphologies of novel linear-hyperbranched and linear brush-like diblock copolymer structure is investigated. An innovative, generally applicable strategy for the preparation of these defined diblock copolymers, consisting of linear polystyrene and branched polycarbosilane blocks, is demonstrated. Furthermore, complete characterization and solid-state morphological studies are provided. Finally, the concept is extended to linear-hyperbrached and linear brush-like polyalkoxysilanes. A shift of the classical phase boundaries to higher PS weight fractions as well as the appearance of new morphologies confirms the dramatic effect that polymer topology has on the morphology of BCPs.

Molekulare Reaktionsmuster in dendritischen Zellen nach Allergenexposition

Allergische Erkrankungen, wie zum Beispiel die allergische Rhinitis oder das allergische Asthma haben im Verlauf der letzten vier Jahrzehnte stark zugenommen. So leidet heute jeder vierte bis fünfte Mensch an einer Allergie. Ausgelöst wird diese IgE-vermittelte Hypersensibilitätsreaktion des Typs I (Allergie vom Soforttyp) von Allergenen und beruht auf der Aktivierung von Mastzellen durch die Interaktion eines Antigens mit dem an eine Mastzelle über die Fc-Rezeptoren gebundenen IgE-Moleküls. Die degranulierende Mastzelle sezerniert Mediatoren, was zu einem Auftreten von allergischen Symptomen führt. Die Bildung von IgE wird durch das von TH2-Zellen produzierte Zytokin IL-4 induziert. Das von TH1-Zellen produzierte Zytokin IFN- ist in der Lage die Sekretion von IL-4 zu inhibieren, wie auch IL-4 hemmend auf die Produktion von IFN- wirkt. Dieses TH1-/ TH2-Gleichgewicht ist bei allergischen Erkrankungen in Richtung TH2 verschoben. Allergene werden von antigenpräsentierenden Zellen aufgenommen, prozessiert und auf der Zelloberfläche präsentiert. Die potentesten antigenpräsentierenden Zellen sind die dendritischen Zellen, die nach Kontakt mit einem Allergen in die benachbarten Lymphknoten wandern, ausreifen und kostimulatorische Moleküle exprimieren. Sie sind so in der Lage T-Zellen zu aktivieren und entweder in TH1- oder in TH2-Zellen differenzieren zu lassen. Die zytokinabhängige TH1- beziehungsweise TH2-Differenzierung führt zur Aktivierung der Januskinasen. Im aktiven Zustand phosphorylieren sie STAT-Moleküle, die dimerisieren und in den Zellkern translozieren, wo sie unter anderem als Transkriptionsfaktoren für Zytokingene dienen. Unreife humane dendritische Zellen von Allergikern zeigen nach Stimulation mit Proteinallergenen eine schnelle Phosphorylierung des mit der TH2-Entwicklung assoziierten STAT6. Dahingegen sind TH1-Antwort hervorrufende Kontaktallergene nicht in der Lage STAT6 oder andere STAT-Moleküle in dendritischen Zellen zu induzieren. Die Transkriptionsfaktoren T-bet und GATA3 sind ebenfalls von Bedeutung für die TH1-/TH2-Entwicklung, da T-bet ausschließlich in TH1-Zellen, GATA3 nur in TH2-Zellen exprimiert wird. Die Regulation des JAK/STAT-Weg unterliegt den Molekülen der intrazellulär vorkommenden Familie der SOCS-Proteine. SOCS3 ist in TH2-Zellen höher exprimiert als SOCS1, wohingegen SOCS1 in TH1-Zellen eine erhöhte Expression gegenüber SOCS3 aufweist. In der vorliegenden Arbeit wurde der Einfluss von Proteinallergenen auf humane dendritische Zellen untersucht. Zunächst konnte eine morphologische Veränderung der unreifen dendritischen Zellen nach Kontakt mit dem Allergenextrakt beobachtet werden. Die beginnende Ausreifung der Zellen konnte mittels Durchflußzytometrie anhand der kostimulatorischen Moleküle CD80 und CD86, insbesondere aber über den Marker für reife dendritische Zellen CD83, nachgewiesen werden. Die zu beobachtende beginnende Ausreifung scheint ein Effekt des bakteriellen Lipopolysaccharids (LPS) zu sein, das in dem Allergenextrakt vorkommt, da sich durch Zugabe des kationischen Antibiotikums Polymyxin B die beginnende Reifung verhindern ließ. Auf RNA-Ebene war es im Rahmen dieser Arbeit möglich, den Einfluss verschiedener Allergene auf unreifen humanen dendritischen Zellen näher zu charakterisieren. So weisen unreife humane dendritische Zellen nach Kontakt mit Proteinallergenextrakt ein TH2-assoziiertes Genexpressionprofil auf, was sich durch eine erhöhte relative Expression der Gene SOCS3 und GATA3 auszeichnet. Im Gegensatz hierzu zeigen unreife humane dendritische Zellen nach Inkubation mit dem Kontaktallergen MCI/MI eine erhöhte relative Expression des Gens T-bet, was mit einer TH1-Antwort assoziiert ist. Nach Zugabe des „TH1-/ TH2-neutralen“ Tetanustoxoids konnten erhöhte relative Expressionen der Gene GATA3, T-bet und SOCS3 gemessen werden. Die Ergebnisse in dem in dieser Arbeit benutzten humanen in vitro System geben Anlass zur Hypothese, dass die Art der Immunantwort (TH1 versus TH2) sich bereits auf Ebene der dendritischen Zellen anbahnt. GeneChip-Analysen mittels High Density Micro Arrays von unreifen humanen dendritischen Zellen, die entweder mit Proteinallergenextrakt oder mit LPS in Berührung kamen, zeigten statistisch signifikant regulierte Gene, die allerdings keine Gemeinsamkeiten aufwiesen. Es konnten für die mit Alllergenextrakt gepulsten dendritischen Zellen insgesamt 10 Gene identifiziert werden, jedoch gelang es nicht, diese näher zu deuten oder in einen Zusammenhang mit der allergischen Erkrankung oder der dendritischen Zelle zu bringen. Für die mit LPS, dem stärkeren Stimulus, gepulsten dendritischen Zellen konnten 40 Gene identifiziert werden, die unter anderem für die Maturierung der dendritischen Zelle verantwortlich sind. Zudem war es möglich, die Daten der Arrays auf Proteinebene exemplarisch anhand des Chemokins CXCL2 (Gro-β) zu verifizieren.

Detektion neuroaktiver Substanzen durch Modifikation des Aktivitätsmusters neokortikaler Netzwerke auf Multielektrodenarrays – Charakterisierung und Optimierung eines neuronalen Biosensors

In dieser Arbeit wurden kortikale neuronale Netzwerke auf Multielektrodenarrays auf ihre Tauglichkeit als zellbasiertes Biosensorsystem untersucht. Der Schwerpunkt der pharmakologischen Untersuchungen an den ausgereiften kortikalen Netzwerken lag auf dem Einsatz von Substanzen, welche auf den GABAA-Rezeptor einwirken. Die Modifikation des spontan generierten Aktivitätsmusters ließ dabei Rückschlüsse auf die Wirksamkeit und den Wirkungsmechanismus der Testsubstanzen zu. Ferner war in den meisten Fällen eine Diskriminierung der auf den gleichen Rezeptor einwirkenden Substanzen möglich. Die Analyse der Spikerate und verschiedener auf Bursts beruhender Messparameter machte deutlich, dass die Burstrate bei den extrazellulären Ableitungen auf Netzwerkebene den sensitivsten und verlässlichsten Parameter zum Nachweis der Substanzeffekte darstellte. Durch die Verwendung kortikaler Netzwerke unter optimierten Kulturbedingungen und einer auf das System abgestimmten Analysesoftware konnte die Reproduzierbarkeit und Sensitivität im Vergleich zu anderen Studien deutlich verbessert werden. Um die extrazelluläre Signalableitung von einer möglichst geringen Zellanzahl und damit einem überschaubaren zellulären Netzwerk auf Multielektrodenarrays zu ermöglichen, wurden die Oberflächeneigenschaften der Substrate so modifiziert, dass die Lokalisation der Zellsomata und das Auswachsen der Neurite einer geometrischen Kontrolle unterlag. Die kontrollierte Substratbeschichtung des Adhäsionspromotors Poly-D-Lysin in einem triangulären Muster konnte dabei durch die Methode des Mikrokontaktstempelns realisiert werden. Durch das kontrollierte Zellwachstum konnte die extrazelluläre Ableitung von Netzwerken einer geringen Zelldichte über einen Zeitraum von mehreren Wochen ermöglicht werden. Die Untersuchung struktureller und morphogenetischer Eigenschaften, sowie elektrophysiologische Untersuchungen der strukturierten Netzwerke bewiesen, dass die kontrollierte Substratbeschichtung sich nicht negativ auf das Wachstum, die Synaptogenese und die Funktionalität auswirkte.

Development of an experiment for ultrahigh-precision g-factor

Die vorliegende Arbeit befasst sich mit der Entwicklung und dem Aufbau eines Experiments zur hochpräzisen Bestimmung des g-Faktors gebundener Elektronen in hochgeladenen Ionen. Der g-Faktor eines Teilchens ist eine dimensionslose Konstante, die die Stärke der Wechselwirkung mit einem magnetischen Feld beschreibt. Im Falle eines an ein hochgeladenes Ion gebundenen Elektrons, dient es als einer der genausten Tests der Quantenelektrodynamik gebundener Zustande (BS-QED). Die Messung wird in einem dreifach Penning-Fallen System durchgeführt und basiert auf dem kontinuierlichen Stern-Gerlach-Effekt. Der erste Teil dieser Arbeit gibt den aktuellen Wissensstand über magnetische Momente wieder. Der hier gewählte experimentelle Aufbau wird begründet. Anschließend werden die experimentellen Anforderungen und die verwendeten Messtechniken erläutert. Das Ladungsbrüten der Ionen - einer der wichtigsten Aufgaben dieser Arbeit - ist dargestellt. Seine Realisierung basiert auf einer Feld-Emissions-Spitzen-Anordnung, die die Messung des Wirkungsquerschnitts für Elektronenstoßionisation ermöglicht. Der letzte Teil der Arbeit widmet sich der Entwicklung und dem Aufbau des Penning-Fallen Systems, sowie der Implementierung des Nachweisprozesses. Gegenwärtig ist der Aufbau zur Erzeugung hochgeladener Ionen und der dazugehörigen Messung des g-Faktors abgeschlossen, einschließlich des Steuerprogramms für die erste Datennahme. Die Ionenerzeugung und das Ladungsbrüten werden die nächsten Schritte sein.

Polymer properties measured by micromechanical cantilever technique

Surface stress changes induced by specific adsorption of molecules were investigated using a micromechanical cantilever sensor (MCS) device. 16 MCS are grouped within four separate wells. Each well can be addressed independently by different liquid enabling functionalization of MCS separately by flowing different solutions through each well and performing sensing and reference experiments simultaneously. In addition, each well contains a fixed reference mirror, which allows measuring the absolute bending of MCS. The effect of the flow rate on the MCS bending change was found to be dependent on the absolute bending value of MCS. In addition, the signal from the reference mirror can be used to follow refractive index changes upon mixing different solutions. Finite element simulation of solution exchange in wells was compared with experiment results. Both revealed that one solution can be exchanged by another one after a total volume of 200 µl has flown through. Using MCS, the adsorption of thiolated deoxyribonucleic acid (DNA) molecules and 6-mercapto-1-hexanol (MCH) on gold surfaces, and the DNA hybridization were performed. The nanomechanical response is in agreement with data reported by Fritz et al.1 Thus, the multiwell device is readily applicable for sensing of multiple chemical and biological recognition events in a single step. In this context controlled release and uptake of drugs are currently widely discussed. As a model system, we have used polystyrene (PS) spheres with diameters in the order of µm. The swelling behavior of individual PS spheres in toluene vapor was studied via mass loading by means of micromechanical cantilever sensors. For 4–8% cross-linked PS a mass increase of 180% in saturated toluene vapor was measured. In addition, the diameter change in saturated toluene vapor was measured and the corresponding volume increase of 200% was calculated. The mass of the swollen PS sphere decreases with increasing exposure time to ultraviolet (UV) light. The swelling response is significantly different between the first and the second exposure to toluene vapor. This is attributed to the formation of a cross-linked shell at the surface of the PS spheres. Shape persistent parts were observed for locally UV irradiated PS spheres. These PS spheres were found to be fluorescent and cracks occur after exposure in toluene liquid. The diffusion time of dye molecules in PS spheres increases with increasing chemical cross-linking density. This concept of locally dissolving non cross-linked PS from the sphere was applied to fabricate donut structures on surfaces. Arrays of PS spheres were fabricated using spin coating. The donut structure was produced simply after liquid solvent rinsing. The complete cross-linking of PS spheres was found after long exposure time to UV. We found that stabilizers play a major role in the formation of the donut nanostructures.

Neurons derived from P19 embryonic carcinoma cells as a platform for biosensor applications - optimisation and characterisation

P19 is a mouse-derived embryonal carcinoma cell line capable of differentiation toward ectodermal, mesodermal and endodermal lineages and could thus be differentiated into neurons. Different culture conditions were tested to optimise and increase the efficiency of neuronal differentiation since the population of P19-derived neurons was reported to be heterogeneous with respect to the morphology and neurotransmitters they synthesise. P19-derived neurons were cultured on microelectrode arrays as cell aggregates and as dissociated cells. Improved neuronal maturation was shown by the presence of microtubule associated protein 2, neurofilament and synaptophysin formation when initiation of neuronal differentiation was prolonged. High initial cell density cultures and coating of surfaces with polyethylenimine-laminin further improved neuronal maturation of differentiated P19 cells. Increased spontaneous activities of the P19-derived neurons were correspondingly recorded. Two to three hours recordings were performed between 17 and 25 days when extracellular signals were stabilised. It was found that P19-derived neurons developed network properties as partially synchronised network activities. P19-derived neurons appeared to give inhomogenous response to the 2 major neurotransmitters, -aminobutyric acid (GABA) and glutamate. The P19-derived neuronal networks obtained from optimised protocol in this thesis were predominantly GABAergic. The reproducible long term extracellular recordings performed showed that neurons derived from P19 embryonal carcinoma cells could be applied as a model for cell based biosensor in corporation with microelectrode arrays.

Surface-plasmon optical and electrochemical characterization of biofunctional surface architectures

The development and characterization of biomolecule sensor formats based on the optical technique Surface Plasmon Resonance (SPR) Spectroscopy and electrochemical methods were investigated. The study can be divided into two parts of different scope. In the first part new novel detection schemes for labeled targets were developed on the basis of the investigations in Surface-plamon Field Enhanced Spectroscopy (SPFS). The first one is SPR fluorescence imaging formats, Surface-plamon Field Enhanced Fluorescence Microscopy (SPFM). Patterned self assembled monolayers (SAMs) were prepared and used to direct the spatial distribution of biomolecules immobilized on surfaces. Here the patterned monolayers would serve as molecular templates to secure different biomolecules to known locations on a surface. The binding processed of labeled target biomolecules from solution to sensor surface were visually and kinetically recorded by the fluorescence microscope, in which fluorescence was excited by the evanescent field of propagating plasmon surface polaritons. The second format which also originates from SPFS technique, Surface-plamon Field Enhanced Fluorescence Spectrometry (SPFSm), concerns the coupling of a fluorometry to normal SPR setup. A spectrograph mounted in place of photomultiplier or microscope can provide the information of fluorescence spectrum as well as fluorescence intensity. This study also firstly demonstrated the analytical combination of surface plasmon enhanced fluorescence detection with analyte tagged by semiconducting nano- crystals (QDs). Electrochemically addressable fabrication of DNA biosensor arrays in aqueous environment was also developed. An electrochemical method was introduced for the directed in-situ assembly of various specific oligonucleotide catcher probes onto different sensing elements of a multi-electrode array in the aqueous environment of a flow cell. Surface plasmon microscopy (SPM) is utilized for the on-line recording of the various functionalization steps. Hybridization reactions between targets from solution to the different surface-bound complementary probes are monitored by surface-plasmon field-enhanced fluorescence microscopy (SPFM) using targets that are either labeled with organic dyes or with semiconducting quantum dots for color-multiplexing. This study provides a new approach for the fabrication of (small) DNA arrays and the recording and quantitative evaluation of parallel hybridization reactions. In the second part of this work, the ideas of combining the SP optical and electrochemical characterization were extended to tethered bilayer lipid membrane (tBLM) format. Tethered bilayer lipid membranes provide a versatile model platform for the study of many membrane related processes. The thiolipids were firstly self-assembled on ultraflat gold substrates. Fusion of the monolayers with small unilamellar vesicles (SUVs) formed the distal layer and the membranes thus obtained have the sealing properties comparable to those of natural membranes. The fusion could be monitored optically by SPR as an increase in reflectivity (thickness) upon formation of the outer leaflet of the bilayer. With EIS, a drop in capacitance and a steady increase in resistance could be observed leading to a tightly sealing membrane with low leakage currents. The assembly of tBLMs and the subsequent incorporation of membrane proteins were investigated with respect to their potential use as a biosensing system. In the case of valinomycin the potassium transport mediated by the ion carrier could be shown by a decrease in resistance upon increasing potassium concentration. Potential mediation of membrane pores could be shown for the ion channel forming peptide alamethicin (Alm). It was shown that at high positive dc bias (cis negative) Alm channels stay at relatively low conductance levels and show higher permeability to potassium than to tetramethylammonium. The addition of inhibitor amiloride can partially block the Alm channels and results in increase of membrane resistance. tBLMs are robust and versatile model membrane architectures that can mimic certain properties of biological membranes. tBLMs with incorporated lipopolysaccharide (LPS) and lipid A mimicking bacteria membranes were used to probe the interactions of antibodies against LPS and to investigate the binding and incorporation of the small antimicrobial peptide V4. The influence of membrane composition and charge on the behavior of V4 was also probed. This study displays the possibility of using tBLM platform to record and valuate the efficiency or potency of numerous synthesized antimicrobial peptides as potential drug candidates.

Noble-metal nanoparticles produced with colloidal lithography: fabrication, optical properties and applications

In this work, metal nanoparticles produced by nanosphere lithography were studied in terms of their optical properties (in connection to their plasmon resonances), their potential application in sensing platforms - for thin layer sensing and bio-recognition events -, and for a particular case (the nanocrescents), for enhanced spectroscopy studies. The general preparation procedures introduced early in 2005 by Shumaker-Parry et al. to produce metallic nanocrescents were extended to give rise to more complex (isolated) structures, and also, by combining colloidal monolayer fabrication and plasma etching techniques, to arrays of them. The fabrication methods presented in this work were extended not only to new shapes or arrangements of particles, but included also a targeted surface tailoring of the substrates and the structures, using different thiol and silane compounds as linkers for further attachment of, i.e. polyelectrolyte layers, which allow for a controlled tailoring of their nanoenvironment. The optical properties of the nanocrescents were studied with conventional transmission spectroscopy; a simple multipole model was adapted to explain their behaviour qualitatively. In terms of applications, the results on thin film sensing using these particles show that the crescents present an interesting mode-dependent sensitivity and spatial extension. Parallel to this, the penetrations depths were modeled with two simplified schemes, obtaining good agreement with theory. The multiple modes of the particles with their characteristic decay lengths and sensitivities represent a major improvement for particle-sensing platforms compared to previous single resonance systems. The nanocrescents were also used to alter the emission properties of fluorophores placed close to them. In this work, green emitting dyes were placed at controlled distances from the structures and excited using a pulsed laser emitting in the near infrared. The fluorescence signal obtained in this manner should be connected to a two-photon processes triggered by these structures; obtaining first insight into plasmon-mediated enhancement phenomena. An even simpler and faster approach to produce plasmonic structures than that for the crescents was tested. Metallic nanodiscs and nanoellipses were produced by means of nanosphere lithography, extending a procedure reported in the literature to new shapes and optical properties. The optical properties of these particles were characterized by extinction spectroscopy and compared to results from the literature. Their major advantage is that they present a polarization-dependent response, like the nanocrescents, but are much simpler to fabricate, and the resonances can be tailored in the visible with relative ease. The sensing capabilities of the metallic nanodiscs were explored in the same manner as for the nanocrescents, meaning their response to thin layers and to bio-recognition events on their surface. The sensitivity of these nanostructures to thin films proved to be lower than that of the crescents, though in the same order of magnitude. Experimental information about the near field extension for the Au nanodiscs of different sizes was also extracted from these measurements. Further resonance-tailoring approaches based on electrochemical deposition of metals on the nanodiscs were explored, as a means of modifying plasmon resonances by changing surface properties of the nanoparticles. First results on these experiments would indicate that the deposition of Ag on Au on a submonolayer coverage level can lead to important blue-shifts in the resonances, which would open a simple way to tailor resonances by changing material properties in a local manner.

Complex materials via colloidal crystallization

The work presented in this thesis deals with complex materials, which were obtained by self-assembly of monodisperse colloidal particles, also called colloidal crystallization. Two main fields of interest were investigated, the first dealing with the fabrication of colloidal monolayers and nanostructures, which derive there from. The second turned the focus on the phononic properties of colloidal particles, crystals, and glasses. For the fabrication of colloidal monolayers a method is introduced, which is based on the sparse distribution of dry colloidal particles on a parent substrate. In the ensuing floating step the colloidal monolayer assembles readily at the three-phase-contact line, giving a 2D hexagonally ordered film under the right conditions. The unique feature of this fabrication process is an anisotropic shrinkage, which occurs alongside with the floating step. This phenomenon is exploited for the tailored structuring of colloidal monolayers, leading to designed hetero-monolayers by inkjet printing. Furthermore, the mechanical stability of the floating monolayers allows the deposition on hydrophobic substrates, which enables the fabrication of ultraflat nanostructured surfaces. Densely packed arrays of crescent shaped nanoparticles have also been synthesized. It is possible to stack those arrays in a 3D manner allowing to mutually orientate the individual layers. In a step towards 3D mesoporous materials a methodology to synthesize hierarchically structured inverse opals is introduced. The deposition of colloidal particles in the free voids of a host inverse opal allows for the fabrication of composite inverse opals on two length scales. The phononic properties of colloidal crystals and films are characterized by Brillouin light scattering (BLS). At first the resonant modes of colloidal particles consisting of polystyrene, a copolymer of methylmethacrylate and butylacrylate, or of a silica core-PMMA shell topography are investigated, giving insight into their individual mechanical properties. The infiltration of colloidal films with an index matching liquid allows measuring the phonon dispersion relation. This leads to the assignment of band gaps to the material under investigation. Here, two band gaps could be found, one originating from the fcc order in the colloidal crystal (Bragg gap), the other stemming from the vibrational eigenmodes of the colloidal particles (hybridization gap).