Herz-Kreislauf-Erkrankungen sind weltweit die häufigste Todesursache. Im Falle eines akuten myokardialen Infarkts ist eine schnelle und präzise Diagnostik notwendig, um ggf. die entsprechend überlebenswichtigen Maßnahmen in die Wege zu leiten. Einer der wichtigsten Biomarker für die Herzinfarkt-Diagnostik ist das kardiale Troponin I (cTnI), welches durch das Absterben der Myokardzellen (Herzmuskel) in den Blutkreislauf gelangt und sich dessen Konzentration über die Zeit ändert. Diese Konzentrationsänderung ist entscheidend für die Diagnostik und letztendlich für weitere lebensrettende medizinische Schritte. Entsprechend wurden in dieser Arbeit verschiedene Messmethoden entwickelt, um cTnI-Konzentrationen zu bestimmen. Zunächst wurden mehrere Enzyme-linked Immunosorbent Assays (ELISA) im Sandwich-Format entwickelt, um eine höchstmögliche Sensitivität zur Detektion von cTnI zu ermöglichen. Um eine schnelle und kostengünstige Detektion von cTnI zu ermöglichen, wurde außerdem ein Lateral-Flow-Assay (LFA) entwickelt. Dabei wurden Gold-Nanoshells für die optische Auswertung verwendet, welche eine empfindlichere Detektion als übliche Gold-Nanopartikel ermöglichen. Des Weiteren wurden verschiedene Immunisierungen mit unterschiedlichen Strategien durchgeführt, um neue Antikörper gegen humanes cTnI zu entwickeln. Für das Online-Monitoring von cTnI wurde der Prototyp eines Biosensors entwickelt, welcher auf Chemilumineszenz-Detektion basiert. Für cTnI wurde mit dem Biosensor eine Nachweisgrenze von 0,6 μg/L (25 pM) in Puffer, 1,8 μg/L (73 pM) in Serum und 1,5 μg/L (63 pM) erzielt. / Cardiovascular diseases are the most common cause of death worldwide. In the event of an acute myocardial infarction, rapid and precise diagnostics are necessary in order to initiate the appropriate vital measures if necessary. One of the most important biomarkers for heart attack diagnostics is cardiac troponin I (cTnI), which enters the bloodstream when the myocardial cells (heart muscle) undergo necrosis after an infarction and whose concentration changes over time. This change in concentration is crucial for diagnostics and ultimately for further lifesaving medical steps. Accordingly, various measurement methods were developed in this work to determine cTnI concentrations. Initially, several enzyme-linked immunosorbent assays (ELISA) were developed in a sandwich format to enable the highest possible sensitivity for the detection of cTnI. A lateral flow assay (LFA) was also developed to enable rapid and cost-effective detection of cTnI. Gold nanoshells were used for optical evaluation, which enable more sensitive detection than conventional gold nanoparticles. Furthermore, different immunizations with different strategies were performed to develop new antibodies against human cTnI. A prototype biosensor based on chemiluminescence detection was developed for the online monitoring of cTnI. For cTnI, a detection limit of 0.6 μg/L (25 pM) in buffer, 1.8 μg/L (73 pM) in serum and 1.5 μg/L (63 pM) was achieved with the biosensor.

The reliable quantitative detection of biomarkers and pathogens at picomolar or even lower concentration would be a great help in point-of-care testing but is not readily available today. Integrated optical waveguides, which interact with the biochemical species to be monitored, are promising candidates for the detection of such ultra-low concentrations. The focus of this thesis is on optical waveguides in the shape of micro-ring or micro-racetrack resonators that are manufactured by UV-assisted nanoimprint lithography. This replica manufacturing technology is analyzed using analytical and numerical models in order to identify and quantify the main influence factors that determine the limit of detection of such biosensors. Potential biosensor applications are evaluated and general design rules are derived. The resulting measurements confirm the high potential of the chosen approach with respect to excellent sensitivity, low limit of detection and high dynamic range. With suitable optimization of the sensor layout, a further improvement of the performance by one to two orders of magnitude is possible.:Editor’s Preface Variables and constants Abbreviations 1 Introductions 1.1 Medical laboratory diagnostics 1.2 Biosensor technologies for point-of-care testing 1.3 Integrated optical waveguides and microresonators 1.4 Outline of the thesis 2 Basics 2.1 Guided waves in planar optical waveguides 2.1.1 Planar optical waveguides 2.1.2 Propagation of optical waves 2.1.3 Coupled modes in waveguides 2.2 Planar optical microresonators 2.2.1 Basic layouts and parameters 2.2.2 Manufacturing 2.2.3 Biosensing 2.3 Functionalization and biofunctionalization 3 UV-NIL Polymer Microresonator Biosensor Design 3.1 UV-assisted nanoimprint lithography 3.2 Waveguide cross-sections and refractive indices 3.2.1 Analytical waveguide modeling 3.2.2 Mode diagrams 3.2.3 Conclusions 3.3 Waveguide coupling 3.4 Waveguide losses 3.4.1 Absorption loss 3.4.2 Roughness loss 3.4..3 Substrate loss 3.4.4 Radiation loss due to bending 3.5 Sensitivity of the effective index to analyte binding 3.6 Overall sensitivity and detection limit 3.7 Generic design guidelines 3.8 Parameter selection for UV-NIL polymer waveguides 3.9 Comparison of polymer and silicon-based waveguides 3.9.1 Waveguide geometry 3.9.2 Radiation loss due to bending 3.9.3 Material damping 3.9.4 Surface roughness 3.9.5 Coupling channel widths and coupling coefficients 3.9.6 Conclusions 4 Characterization and Proof of Concept 4.1 Manufacturing-based design limits and chosen designs 4.2 Measurement setup and characterization process 4.3 Optical properties of UV-NIL polymer microresonators 4.4 Proof of concept 4.4.1 Sensitivity to bulk solutions 4.4.2 Reproducibility and drift 4.4.3 Comparison with theory 4.4.4 Comparison with literature 4.4.5 Sensitivity improvement 4.5 Asymmetry of the resonance curves 4.5.1 Cavity lifetime 4.5.2 Thermal influence 4.5.3 Summary 4.6 Conclusions 5 Integration into a biosensor platform 5.1 Chemical functionalization by oxygen plasma 5.2 Preparation of a biosensor characterization assay 5.2.1 Binding of fluorescent nanoparticles onto polymer surfaces 5.3 Microfluidic system 5.3.1 Programmable microfluidic system 5.3.2 System evaluation and improvement 5.4 Conclusions 6 Conclusions Declaration of authorship Acknowledgements Publications and awards / Der zuverlässige quantitative Nachweis von Biomarkern und Krankheitserregern in pikomolarer oder noch niedrigerer Konzentration wäre eine große Hilfe bei Tests am Point-of-Care, ist aber heute nicht ohne weiteres verfügbar. Integrierte optische Wellenleiter, die mit den zu überwachenden biochemischen Spezies interagieren, sind vielversprechende Kandidaten für den Nachweis solcher ultraniedriger Konzentrationen. Der Schwerpunkt dieser Arbeit liegt auf optischen Wellenleitern in Form von Mikro-Ring- oder Mikro-Spur-Resonatoren, die durch UV-unterstützte Nanoimprint-Lithographie hergestellt werden. Diese Replika-Herstellungstechnologie wird mit Hilfe analytischer und numerischer Modelle analysiert, um die wichtigsten Einflussfaktoren zu identifizieren und zu quantifizieren, die die Nachweisgrenze solcher Biosensoren bestimmen. Potenzielle Biosensoranwendungen werden bewertet und allgemeine Designregeln abgeleitet. Die daraus resultierenden Messungen bestätigen das hohe Potenzial des gewählten Ansatzes in Bezug auf ausgezeichnete Empfindlichkeit, niedrige Nachweisgrenze und hohen Dynamikbereich. Bei geeigneter Optimierung des Sensorlayouts ist eine weitere Verbesserung der Leistung um ein bis zwei Größenordnungen möglich.:Editor’s Preface Variables and constants Abbreviations 1 Introductions 1.1 Medical laboratory diagnostics 1.2 Biosensor technologies for point-of-care testing 1.3 Integrated optical waveguides and microresonators 1.4 Outline of the thesis 2 Basics 2.1 Guided waves in planar optical waveguides 2.1.1 Planar optical waveguides 2.1.2 Propagation of optical waves 2.1.3 Coupled modes in waveguides 2.2 Planar optical microresonators 2.2.1 Basic layouts and parameters 2.2.2 Manufacturing 2.2.3 Biosensing 2.3 Functionalization and biofunctionalization 3 UV-NIL Polymer Microresonator Biosensor Design 3.1 UV-assisted nanoimprint lithography 3.2 Waveguide cross-sections and refractive indices 3.2.1 Analytical waveguide modeling 3.2.2 Mode diagrams 3.2.3 Conclusions 3.3 Waveguide coupling 3.4 Waveguide losses 3.4.1 Absorption loss 3.4.2 Roughness loss 3.4..3 Substrate loss 3.4.4 Radiation loss due to bending 3.5 Sensitivity of the effective index to analyte binding 3.6 Overall sensitivity and detection limit 3.7 Generic design guidelines 3.8 Parameter selection for UV-NIL polymer waveguides 3.9 Comparison of polymer and silicon-based waveguides 3.9.1 Waveguide geometry 3.9.2 Radiation loss due to bending 3.9.3 Material damping 3.9.4 Surface roughness 3.9.5 Coupling channel widths and coupling coefficients 3.9.6 Conclusions 4 Characterization and Proof of Concept 4.1 Manufacturing-based design limits and chosen designs 4.2 Measurement setup and characterization process 4.3 Optical properties of UV-NIL polymer microresonators 4.4 Proof of concept 4.4.1 Sensitivity to bulk solutions 4.4.2 Reproducibility and drift 4.4.3 Comparison with theory 4.4.4 Comparison with literature 4.4.5 Sensitivity improvement 4.5 Asymmetry of the resonance curves 4.5.1 Cavity lifetime 4.5.2 Thermal influence 4.5.3 Summary 4.6 Conclusions 5 Integration into a biosensor platform 5.1 Chemical functionalization by oxygen plasma 5.2 Preparation of a biosensor characterization assay 5.2.1 Binding of fluorescent nanoparticles onto polymer surfaces 5.3 Microfluidic system 5.3.1 Programmable microfluidic system 5.3.2 System evaluation and improvement 5.4 Conclusions 6 Conclusions Declaration of authorship Acknowledgements Publications and awards

When exposed to light, metal nanoparticles exhibit a phenomenon known as LSPR, Localized Surface Plasmon Resonance. The wavelengths at which LSPR occurs is very dependent on the refractive index of the surrounding medium. Binding of biomolecules to the surface of gold nanoparticles result in a change in the refractive index that can be detected spectrophotometrically by monitoring the LSPR peak shift. When functionalized with the corresponding ligand(s), gold nanoparticles can be utilized in biosensors to detect the presence and concentration of a predetermined analyte. However, the system must exhibit high specificity and give rise to a detectable shift for analytes in the desired concentration range to be of commercial interest. The aim of the diploma project was to investigate and optimize the biofunctionalization and performance of a fiber optics based LSPR biosensor.  Three ligand systems were investigated for detection of antibodies (IgG), insulin and avidin. Binding of the analyte to the ligand caused a shift of a few nanometers when using spherical gold nanoparticles. The shifts were significantly larger when using gold nanorods. When using the IgG and insulin ligands, only minor unspecific binding was observed. The setup thus shows great potential for use in a wide range of sensing applications.

A contactless method using a novel design of the experimental cell for formation of porous silicon with morphological gradient is reported. Fabricated porous silicon layers show a large distribution in porosity, pore size and depth along the radius of the samples. Symmetrical arrangements of morphology gradient were successfully formulated radially on porous films and the formation was attributed to decreasing current density radially inward on the silicon surface exposed to Triton (R) X-100 containing HF based etchant solution. Increasing the surfactant concentration increases the pore depth gradient but has a reverse effect on the pore size distribution. Interestingly, when dimethyl sulfoxide was used instead of Triton (R) X-100 in the etchant solution, no such morphological gradients were observed and a homogeneous porous film was formed.

Double-exposure lithography has shown promise as potential resolu- tion enhancement technique that is attractive because it is much cheaper than double-patterning lithography and it can be deployed on existing imaging tools. However, this technology is not possible without the development of new materials with nonlinear response to exposure dose. Several materials have been proposed to implement a nonlinear response to exposure including re- versible contrast enhancement layers (rCELs), two-photon materials, interme- diate state two-photon (ISTP) materials, and optical threshold layers (OTLs). The performance of these materials in double-exposure applications was inves- tigated through computer simulation using a custom simulator. The results from the feasibility studies revealed that the ISTP and OTL types of materials showed much more promise than the rCEL and two-photon types of materi- als. Calculations show that two-photon materials will not be feasible unless achievable laser peak power in exposure tools can be signi¯cantly increased. Although rCEL materials demonstrated nonlinear behavior in double-exposure mode, only marginal image quality and process window improvements were ob- served. Using the results from the simulation work described herein, materials development work is currently ongoing to enable potential ISTP and OTL materials for manufacturing. A new biochip platform named \Mesoscale Unaddressed Functional- ized Features INdexed by Shape" (MUFFINS) was developed in the Willson Research Group at the University of Texas at Austin as a potential method to achieve a new low-cost biosensor system. The platform uses poly(ethylene glycol) hydrogels with bioprobes covalently cross-linked into the matrix for detection. Each sensor is shape-encoded with a unique pattern such that the information of the sensor is associated with the pattern and not its position. Large quantities of individual sensors can be produced separately and then self- assembled to form random arrays. Detection occurs through hybridization of the probes with °uorescently labeled targets. The key designs of the system include parallel batch fabrication using photolithography and self-assembly, in- creased information density using multiplexing, and enhanced shape-encoding with automated pattern recognition. The development of two aspects of the platform { self-assembly mechanics and pattern recognition algorithm, and a demonstration of all the key design elements using a single array are described herein. / text

The objective of this thesis is to introduce and demonstrate a novel magnetic bead detector based on inductive detection at the ferromagnetic resonance (FMR) frequency for use in bio-sensing applications. Detection ability is demonstrated through theoretical arguments, numerical computer simulations, and experimental characterization of micro-fabricated detectors. The detector is composed of two uniplanar rf waveguides (coplanar waveguide and slotline) terminated together at a short-circuit junction, which serves as the sensitive area. Experimental characterization of a micro-fabricated junction gives a signal ranging between 1 microvolt/volt and 12 microvolts/volt, depending on the number of beads at the junction as well spatial distribution of the beads. The locations around the tips of the CPW were shown to be the most sensitive. A more complex rf circuit design was created employing the detection junction, and detection of magnetic beads was successfully shown at rf frequencies around 6 GHz in this configuration. Due to lack of FMR characterization data for magnetic beads in the literature, several varieties of magnetic beads were characterized using a CPW transmission line and custom apparatus to determine FMR properties. Finally, successful detection of magnetic beads was demonstrated in a system-level integration experiment employing the detector junction in combination with microfluidics and bio-chemical surface modifications. / Graduation date: 2010

Worldwide, 3.575 million people die each year from water-related diseases. The water and sanitation crisis claims more lives than any warfare and is predicted to be one of the biggest global challenges of this century. The rapid, accurate detection of viral pathogens from environmental samples is an ongoing and pertinent challenge in biological engineering. Currently employed methods are lacking in either efficiency or specificity. Here we explore a novel method for virus detection and concurrently use this method to learn more about the very early stages of the virus infection process. The method combines Fourier transform infrared (FTIR) spectroscopy, a method of visualizing molecules based on changes in vibration of particles, and mammalian cells as the biosensor. This method is used to detect and investigate viruses from the family picornaviridae, chosen due to their public health burden and their widespread presence in environmental samples, especially water sources. This family includes the Polioviruses, echoviruses and Coxsackieviruses, among others, many of which are human pathogens.The research outlined in this dissertation is aimed at developing and implementing a new cell-based biosensor that combines the advantages of FTIR spectroscopy with the ability of buffalo green monkey kidney (BGMK) cells to sense diverse stimuli, including infective enteroviruses. The goal of developing this biosensor is outlined in the first paper. The second paper focuses on the application of advanced statistical methods to analyze the spectra to discriminate different viral infections in BGMK cells. Finally, we designed a non-reactive metal biochamber to use with attenuated total reflectance-FTIR. This allowed near-continuous acquisition of real-time spectral data for the study of biochemical changes in mammalian cells caused by poliovirus (PV1) infection. This system is capable of tracking changes in cell biochemistry in minute intervals for many hours at a time.This work demonstrates the feasibility of FTIR spectroscopy in combination with the broad sensitivity of mammalian cells for potential use in the detection of infective viruses from environmental samples. We envision this method being extended to high throughput, automated systems to screen for viruses or other toxins in drinking water systems and medical applications.

This work has explored the capability of a mixed film composed of oligonucleotides and oligomers to improve the selectivity for the detection of fully complementary oligonucleotide targets in comparison to partially complementary targets which have one and three base-pair mismatched sites. The intention was to introduce a “matrix isolation” effect on oligonucleotide probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-oligonucleotide and/or oligonucleotide-to-surface interactions. This resulted in a more homogeneous environment for probes, thereby minimizing the dispersity of energetics associated with formation of double-stranded hybrids. The mixed film was constructed by immobilizing pre-synthesized oligonucleotides onto a mixed aminosilane layer and then growing the oligomer portion by surface-initiated atom transfer radical polymerization (ATRP) of 2-hydroxy methacrylate (PHEMA). The performance of the mixed film was compared to films composed of only oligonucleotides in a series of hybridization and melt curve experiments. Surface characterization techniques were used to confirm the growth of the oligomer portion as well as the presence of both oligonucleotides and oligomer components. Polyatomic bismuth cluster ions as sources for time-of-flight secondary ion mass spectrometry experiments could detect both components of the mixed film at a high sensitivity even though the oligomer portion was at least 200-fold in excess. At the various ionic strengths investigated, the mixed films were found to increase the selectivity for fully complementary targets over mismatched targets by increasing the sharpness of melt curves and melting temperature differences (delta Tm) by 2- to 3-fold, and by reducing non-specific adsorption. This resulted in improved resolution between the melt curves of fully and partially complementary targets. A fluorescence lifetime investigation of the Cy3 emission demonstrated that Cy3-labeled oligonucleotide probes experienced a more rigid microenvironment in the mixed films. These experiments demonstrated that a mixed film composed of oligonucleotides and PHEMA can be prepared on silica-based substrates, and that they can improve the selectivity for SNP discrimination compared to conventional oligonucleotide films.

Fragment based drug discovery (FBDD) has been applied to two protease drug targets, MMP-12 and HIV-1 protease. The primary screening and characterization of hit fragments were performed with surface plasmon resonance -technology. Further evaluation of the interaction was done by inhibition studies and in one case with X-ray crystallography. The focus of the two projects was different. Many MMP inhibitors contain a strong zinc chelating group, hydroxamate, interacting with the catalytic zinc atom. This strategy may be the cause for the low specificity of MMP inhibitors. Using FBDD we found a fragment with an unusual strong affinity for MMP-12. An inhibition assay confirmed that it was an inhibitor but indicated a stoichiometry of 2:1. Crystallography data revealed that an adduct of the fragment was bound in the active site, with interactions both with the catalytic zinc and the S1’ pocket. This may present a new scaffold for MMP-12 inhibitors. For HIV-1 protease the focus was on identifying inhibitors not sensitive to current resistance mutations. A fragment library for screening with SPR-technology was designed and used for screening against wild type enzyme and three variants with resistance mutations. Many of the hits were promiscuous but a number of fragments with possible allosteric inhibition mechanism were identified. The temperature dependency of the dissociation rate and reported resistance mutations was studied with thermodynamics. A good, but not perfect correlation was found between resistance and both the dissociation data and the free energy for dissociation compared to data from wild type enzyme. However, the type of mutation also influenced the results. The flap mutation G48V displayed thermodynamic profiles not completely correlating with resistance. It was found that dissociation rate and thermodynamics may complement each other when studying resistance, but only one of them may not be enough.

The development of enzymatic biosensors and enzymatic biofuel cells (EBFCs) has been a significant area of research for decades. Enzymatic catalysis can provide for specific, reliable sensing of target analytes as well as the continuous generation of power from physiologically present fuels. However, the broad implementation of enzyme-based devices is still limited by low operational/storage stabilities and insufficient power densities. Approaches to improving upon these limitations have focused on the optimization of enzyme activity and electron transfer kinetics at enzyme-functionalized electrodes. Currently, such optimization can be performed through enzyme structural engineering, improvement of enzyme immobilization methodologies, and fabrication of advantageous electrode materials to enhance retained enzyme activity density at the electrode surface and electron transfer rates between enzymes and an electrode. In this work, varying electrode materials were studied to produce an increased understanding on the impacts of material properties on resulting biochemical, and electrochemical performances upon enzyme immobilization and an additional method of electroactive enzyme-based optimization was developed through the use of polymer-based protein engineering (PBPE). First, graphene/single-wall carbon nanotube cogels were studied as supports for membrane- and mediator-free EBFCs. The high available specific surface area and porosity of these materials allowed the rechargeable generation of a power density within one order of magnitude of the highest performing glucose-based EBFCs to date. Second, two additional carbon nanomaterial-based electrode materials were fabricated and examined as EBFC electrodes. Graphene-coated single-wall carbon nanotube gels and gold nanoparticle/multi-wall carbon nanotube-coated polyacrylonitrile fiber paddles were utilized as electroactive enzyme supports. The performance comparison of these three materials provided an increased understanding of the impact of material properties such as pore size, specific surface area and material surface curvature on enzyme biochemical and electrochemical characteristics upon immobilization. Third, PBPE techniques were applied to develop enzyme-redox polymer conjugates as a new platform for enzymatic biosensor and EBFC optimization. Poly(N-(3-dimethyl(ferrocenyl) methylammonium bromide)propyl acrylamide) (pFcAc) was grown directly from the surface of glucose oxidase (GOX) through atom-transfer radical polymerization. Utilization of the synthesized GOX-pFcAc conjugates led to a 24-fold increase in current generation efficiency and a 4-fold increase in EBFC power density compared to native GOX. GOX-pFcAc conjugates were further examined as working catalysts in carbon paper-based enzymatic biosensors, which provided reliable and selective glucose sensitivities and allowed a systematic analysis of sources of instability in enzyme-polymer conjugate-based biosensors and EBFCs. The knowledge gained through these studies and the in-depth characterization of an additional layer of optimization capacity using PBPE could potentially enhance the progress of enzymatic biosensor and EBFC development.