Oberflächenplasmonen-artige Resonanzen auf metallischen Gittern

Die Geometrie einer Metalloberfläche bestimmt die Wechselwirkung zwischen der Oberflächenplasmonenresonanz und anderen Energieformen wie Photonen, anderen Oberflächenplasmonen und molekularen Anregungen. In dieser Arbeit wird der Einfluss dieser Effekte auf die optischen Eigenschaften von metallischen Reliefgittern untersucht.Aufgrund von Modellrechungen werden auf tiefen Gitterstrukturen Resonanzen erwartet, die sich als selbstgekoppelte Oberflächenplasmonen interpretieren lassen. Diese Resonanzen wurden aufgrund der Symmetrie des elektromagnetischen Feldes klassifiziert. Es wurden tiefe Gitter mit unterschiedlichen Profilformen hergestellt, die die experimentelle Beobachtung von drei gekoppelten Resonanzen erlauben. Variationen in der Tiefe und Asymmetrie der Gitter sowie in der experimentellen Geometrie zeigen die theoretisch vorausgesagten Effekte.Fluoreszenzfarbstoffe können mit elektromagnetischen Oberflächenresonanzen Energie austauschen. Die Lokalisierung des elektrischen Feldes von gekoppelten Resonanzen führt zu einer Ortsabhängigkeit der Kopplung, die bei 'freien' Oberflächenplasmonen nicht beobachtet wird. Ein theoretisches Modell ist in der Lage, die experimentellen Befunde weitgehend zu beschreiben. Die Ortsabhägigkeit der photochemischen Zerstörungsrate erlaubt die Anwendung dieses Effektes zur Messung von Diffusionsphänomenen in dünnen Filmen.Des weiteren wurde die Polarisationsabhängigkeit der Anregung von Oberflächenplasmonen wurde in konischer Reflexionsgeometrie und die Rolle der Oberflächenplasmonen in der thermisch induzierten Lichtemission untersucht. Ferner wurde eine vereinfachte Auswertungsroutine zur Anwendung von Gittern in der Untersuchung von dünnen dielektrischen Filmen entwickelt.

Strategies for detecting DNA hybridisation using surface plasmon fluorescence spectroscopy

Die Anregung und Emission von Fluorophoren nahe planaren Metalloberflächen und schiefen Gittern wurde mittels Oberflächenplasmonen Fluoreszenz Spektroskopie (SPFS) untersucht. Die Fluorophore konnten durch das evaneszente Plasmonenfeld angeregt und die einzelnen Abregungskanäle identifiziert werden.Die Sensorarchitektur für den Nachweis der Hybridisierung bestand aus auf einer Streptavidin-Matrix immobilisierten unmarkierten Sondensträngen. Cy5 markierte Zielsequenzen wurden aus der Lösung hybridisiert und die Adsorptionskinetiken konnten oberflächensensitiv detektiert werden.Ein neues Detektionsschema für unmarkierte Zielstränge wurde mittels fluoreszenzmarkirten Sondensträngen realisiert. Die Hybridisierung führte zu der Bildung von steifen helikalen Bereichen in der Probe und separierte den Farbstoff von der Metalloberfläche. Reduzierte Fluorezenzlöschung zeigte daher das Hybridisierungsereignis an.Die Verwendung eines potentiellen Förster-Paares zur Detektion von DNA Hybridisierung wurde untersucht. Donor und Akzeptor wurden an Ziel- und Sondenstrang immobilisiert und das Hybridisierungsereignis konnte anhand der Auslöschung der Donor-Fluorezenz nachgewiesen werden.Schließlich wurde der Einsatz von einzelstrangbindenden Proteinen (SSB) zur Steigerung der Sensitivität bezüglich Basenfehlpaarungen betrachtet. Verdrängungsreaktionen zwischen Proteinen und markierten Zielsträngen wurden anhand von SPS und Fluorezenzkinetiken studiert.

Decay of imprinted surface waves in polymers

CONCLUSIONS The focus of this work was the investigation ofanomalies in Tg and dynamics at polymer surfaces. Thethermally induced decay of hot-embossed polymer gratings isstudied using laser-diffraction and atomic force microscopy(AFM). Monodisperse PMMA and PS are selected in the Mwranges of 4.2 to 65.0 kg/mol and 3.47 to 65.0 kg/mol,respectively. Two different modes of measurement were used:the one mode uses temperature ramps to obtain an estimate ofthe near-surface glass temperature, Tdec,0; the other modeinvestigates the dynamics at a constant temperature aboveTg. The temperature-ramp experiments reveal Tdec,0 valuesvery close to the Tg,bulk values, as determined bydifferential scanning calorimetry (DSC). The PMMA of65.0 kg/mol shows a decreased value of Tg, while the PS samples of 3.47 and 10.3 kg/mol (Mw

Grating coupled surface plasmon enhanced fluorescence spectroscopy

Over the last three decades, sensors based on the phenomenon of surface plasmon resonance have proven particularly suitable for real time thin film characterization, gas detection, biomolecular interaction examination and to supplement electrochemical methods. Systems based on prism coupling have been combined with fluorescence detection under the name of surface plasmon fluorescence spectroscopy to increase sensitivity even further. Alternatively, metal gratings can be employed to match photons for plasmon resonance. The real time monitoring of binding reactions not yet been reported in the combination of fluorescence detection and grating coupling. Grating-based systems promise more competitive products, because of reduced operating costs, and offer benefits for device engineering. This thesis is comprised of a comprehensive study of the suitability of grating coupling for fluorescence based analyte detection. Fundamental properties of grating coupled surface plasmon fluorescence spectroscopy are described, as well as issues related to the commercial realization of the method. Several new experimental techniques are introduced and demonstrated in order to optimize performance in certain areas and improve upon capabilities in respect to prism-based systems. Holographically fabricated gratings are characterized by atomic force microscopy and optical methods, aided by simulations and profile parameters responsible for efficient coupling are analyzed. The directional emission of fluorophores immobilized on a grating surface is studied in detail, including the magnitude and geometry of the fluorescence emission pattern for different grating constants and polarizations. Additionally, the separation between the minimum of the reflected intensity and the maximum fluorescence excitation position is examined. One of the key requirements for the commercial feasibility of grating coupling is the cheap and faithful mass production of disposable samples from a given master grating. The replication of gratings is demonstrated by a simple hot embossing method with good reproducibility to address this matter. The in-situ fluorescence detection of analyte immobilization and affinity measurements using grating coupling are described for the first time. The physical factors related to the sensitivity of the technique are assessed and the lower limit of detection of the technique is determined for an exemplary assay. Particular attention is paid to the contribution of bulk fluorophores to the total signal in terms of magnitude and polarization of incident and emitted light. Emission from the bulk can be a limiting factor for experiments with certain assay formats. For that reason, a novel optical method, based on the modulation of both polarization and intensity of the incident beam, is introduced and demonstrated to be capable of eliminating this contribution.

New enhancement strategies for plasmon-enhanced fluorescence biosensors

This thesis investigates metallic nanostructures exhibiting surface plasmon resonance for the amplification of fluorescence signal in sandwich immunoassays. In this approach, an analyte is captured by an antibody immobilized on a plasmonic structure and detected by a subsequently bound fluorophore labeled detection antibody. The highly confined field of surface plasmons originates from collective charge oscillations which are associated with high electromagnetic field enhancements at the metal surface and allow for greatly increased fluorescence signal from the attached fluorophores. This feature allows for improving the signal-to-noise ratio in fluorescence measurements and thus advancing the sensitivity of the sensor platform. In particular, the thesis presents two plasmonic nanostructures that amplify fluorescence signal in devices that rely on epifluorescence geometry, in which the fluorophore absorbs and emits light from the same direction perpendicular to the substrate surface.rnThe first is a crossed relief gold grating that supports propagating surface plasmon polaritons (SPPs) and second, gold nanoparticles embedded in refractive index symmetric environment exhibiting collective localized surface plasmons (cLSPs). Finite-difference time-domain simulations are performed in order to design structures for the optimum amplification of established Cy5 and Alexa Fluor 647 fluorophore labels with the absorption and emission wavelengths in the red region of spectrum. The design takes into account combined effect of surface plasmon-enhanced excitation rate, directional surface plasmon-driven emission and modified quantum yield for characteristic distances in immunoassays. Homebuilt optical instruments are developed for the experimental observation of the surface plasmon mode spectrum, measurements of the angular distribution of surface plasmon-coupled fluorescence light and a setup mimicking commercial fluorescence reading systems in epifluorescence geometry.rnCrossed relief grating structures are prepared by interference lithography and multiple copies are made by UV nanoimprint lithography. The fabricated crossed diffraction gratings were utilized for sandwich immunoassay-based detection of the clinically relevant inflammation marker interleukin 6 (IL-6). The enhancement factor of the crossed grating reached EF=100 when compared to a flat gold substrate. This result is comparable to the highest reported enhancements to date, for fluorophores with relatively high intrinsic quantum yield. The measured enhancement factor excellently agrees with the predictions of the simulations and the mechanisms of the enhancement are explained in detail. Main contributions were the high electric field intensity enhancement (30-fold increase) and the directional fluorescence emission at (4-fold increase) compared to a flat gold substrate.rnCollective localized surface plasmons (cLSPs) hold potential for even stronger fluorescence enhancement of EF=1000, due to higher electric field intensity confinement. cLSPs are established by diffractive coupling of the localized surface plasmon resonance (LSPR) of metallic nanoparticles and result in a narrow resonance. Due to the narrow resonance, it is hard to overlap the cLSPs mode with the absorption and emission bands of the used fluorophore, simultaneously. Therefore, a novel two resonance structure that supports SPP and cLSP modes was proposed. It consists of a 2D array of cylindrical gold nanoparticles above a low refractive index polymer and a silver film. A structure that supports the proposed SPP and cLSP modes was prepared by employing laser interference lithography and the measured mode spectrum was compared to simulation results.rn