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Hybrid plasmonic-photonic structures were introduced as novel platforms for on-chip biochemical sensing and spectroscopy. By appropriate coupling of photonic and plasmonic modes, a hybrid architecture was realized that can benefit from the advantages of integrated photonics such as the low propagation loss, ultra-high Q modes, and robustness, as well as the advantages of nanoplasmonics such as extreme light localization, large sensitivities, and ultra-high field enhancements to bring about unique performance advantages for efficient on-chip sensing. These structures are highly sensitive and can effectively interact with the target biological and chemical molecules. It was shown that interrogation of single plasmonic nanoparticles is possible using a hybrid waveguide and microresonator-based structure, in which light is efficiently coupled from photonic structures to the integrated plasmonic structures. The design, implementation, and experimental demonstration of hybrid plasmonic-photonic structures for lab-on-chip biochemical sensing applications were discussed. The design goal was to achieve novel, robust, highly efficient, and high-throughput devices for on-chip sensing. The sensing scenarios of interest were label-free refractive index sensing and SERS. Nanofabrication processes were developed to realize the hybrid plasmonic-photonic structures. Silicon nitride was used as the material platform to realize the integrated photonic structure, and gold was used to realize plasmonic nanostructures. Special optical characterization setups were designed and implemented to test the performance of these nanoplasmonic and nanophotonic structures. The integration of the hybrid plasmonic-photonic structures with microfluidics was also optimized and demonstrated. The hybrid plasmonic-photonic-fluidic structures were used to detect different analytes at different concentrations. A complete course of research from design, fabrication, and characterization to demonstration of sensing applications was conducted to realize nanoplasmonic and integrated photonic structures. The novel structures developed in this research can open up new potentials for biochemical sensors with advanced on-chip functionalities and enhanced performances.

To truly realize and exploit the extremely powerful information given from surface-enhanced Raman scattering (SERS) spectroscopy, it is critical to develop an understanding of how to design highly sensitive and selective substrates, produce specific and label-free spectra of target analytes, and fabricate long-lasting and in-the-field ready platforms for trace detection applications. The study presented in this dissertation investigated the application of two- and three-dimensional substrates composed of highly-ordered metal nanostructures. These systems were designed to specifically detect target analytes that would enable the trace, label-free, and real-time detection of chemicals and biomolecules. Specifically, this work provides new insight into the required properties for maximizing electromagnetic and chemical Raman enhancement in three-dimensional porous alumina substrates by designing metal nanostructure shape, density, aggregated state, and most importantly aligning the substrate pore size with the excitation wavelength used for plasmonic enhancement leading to the ppb detection of vapor phase hazardous chemicals. A new micropatterned silver nanoparticle substrate fabricated via soft lithography with specific functionalization was developed, which allows the simultaneous analyte and background detection for trace concentrations of the target biomolecule, immunoglobulin G. Also, a novel functionalized SERS hot spot fabrication technique, which utilizes highly specific aptamers as both the mediator for electrostatic assembly of gold nanoframe dimers as well as the biorecognition element for the target, riboflavin, to properly locate the tethered biomolecule within the enhanced region for trace detection, was demonstrated. We suggest that the understanding of SERS phenomena that occur at the interface of nanostructures and target molecules combined with the active functionalization and organization of metal nanostructures and trace detection of analytes discussed in this study can provide important insight for addressing some of the challenges facing the field of SERS sensor design such as high sensitivity and selectivity, reliable and repeatable label-free identification of spectral peaks, and the well-controlled assembly of functional metal nanostructures. This research will have a direct impact on the future application of SERS sensors for the trace detection of target species in chemical, environmental, and biomedical fields through the development of specific design criteria and fabrication processes.

The work reported in this thesis is motivated by the need for wireless powering of a miniaturized implantable device for neurophysiological research and possible clinical applications. The antenna used in such applications must be studied in the context of biological tissue media. In this thesis, we perform a preliminary study of antenna behaviour in the complex tissue environment. Our test cases are the wire dipole antenna chosen for its structural simplicity and the spiral antenna, selected for its wide bandwidth. The simulation tool SEMCAD-X, is based on the Finite-Difference Time-Domain (FDTD) method and is used throughout this work. To have an in-depth understanding of the characteristics of different solvers implemented in SEMCAD-X and relevant for our applications, we first simulate the antenna structures in the free-space region using both SEMCAD-X and HFSS (a Finite-Element Method (FEM) simulation software). The cross-platform comparison between these two simulation tools helps us identify the advantages of using conformal FDTD solver over the conventional staircase FDTD solver in SEMCAD-X. We then embed the antennas in tissue-like non-homogeneous lossy media to observe the terminal voltages induced by an impinging plane-wave. These numerical experiments will help us with the assessment of the following: variations of antenna properties with the in-tissue locations, and more importantly the dependence of the induced voltage on the depth of the implant.

Semiconductor quantum dots (QDs) and gold nanoparticles (AuNPs) have gained tremendous attention in the last decade as a result of their size-dependent spectroscopic properties. These nanoparticles have been a subject of intense study to bridge the gap between macroscopic and atomic behavior, as well as to generate new materials for novel applications in therapeutics, biological sensing, light emitting devices, microelectronics, lasers, and solar cells. One of the most promising areas for the use of these nanoparticles is in biotechnology, where their size-dependent optical properties are harnessed for imaging and sensing applications. However, these nanoparticles, as synthesized, are often not stable in aqueous media and lack simple and reliable means of covalently linking to biomolecules. The focus of this work is to advance the progress of these nanomaterials for biotechnology by synthesizing them, characterizing their optical properties and rendering them water-soluble and functional while maintaining their coveted optical properties. QDs were synthesized by an organometallic chemical procedure that utilizes coordinating solvents to provide brightly luminescent nanoparticles. The optical interactions of these QDs were studied as a function of concentration to identify particle size-dependent optimal concentrations, where scattering and indirection excitation are minimized and the amount light observed per particle is maximized. Both QDs and AuNPs were rendered water-soluble and stable in a broad range of biologically relevant conditions by using a series of ligands composed of dihydrolipoic acid (DHLA) appended to poly(ethylene glycol) methyl ether. By studying the stability of the surface modified AuNPs, we revealed some interesting information regarding the role of the surface ligand on the nanoparticle stability (i.e. solubility in high salt concentration, resistance to dithiothreitol competition and cyanide decomposition). Furthermore, the nanoparticles were functionalized using a series of bifunctional ligands that contain a dithiol group (DHLA) for surface binding, a PEG segment to instill water-solubility and a terminal functional group for easy bioconjugation (i.e. NH 2 , COOH, or biotin). Finally, a sensing application was demonstrated to detect the presence of microbial DNA (unmethlylated CpG) by using Toll-like receptor 9 proteins as the recognition components and the QDs as the transduction elements via Förster Resonance Energy Transfer.

The synthesis and biological sensing applications of novel water soluble poly(para-phenyleneethynylene)s (PPEs) are presented. The ease of synthesis, synthetic variability, and dramatic chromicity of PPEs makes them well suited for biological and sensing applications. Molecular recognition and signal transduction can be achieved by using PPEs as sensory materials. By incorporating biological functional groups (e.g. sugars), PPEs can efficiently detect the presence of toxic heavy metals, proteins, and bacteria through either fluorescence quenching or enhancement. Rapid, precise, and convenient sensory arrays for the detection of biological analytes are possible through the formation of gold nanoparticle-PPE constructs.

A fast and direct surface plasmon resonance (SPR) method for the kinetic analysis of the interactions between peptide antigens and immobilised monoclonal antibodies (mAb) has been established. Protocols have been developed to overcome the problems posed by the small size of the analytes (< 1600 Da). The interactions were well described by a simple 1:1 bimolecular interaction and the rate constants were self-consistent and reproducible. The key features for the accuracy of the kinetic constants measured were high buffer flow rates, medium antibody surface densities and high peptide concentrations. The method was applied to an extensive analysis of over 40 peptide analogues towards two distinct anti-FMDV antibodies, providing data in total agreement with previous competition ELISA experiments.Eleven linear 15-residue synthetic peptides, reproducing all possible combinations of the four replacements found in foot-and-mouth disease virus (FMDV) field isolate C-S30, were evaluated. The direct kinetic SPR analysis of the interactions between these peptides and three anti-site A mAbs suggested additivity in all combinations of the four relevant mutations, which was confirmed by parallel ELISA analysis. The four-point mutant peptide (A15S30) reproducing site A from the C-S30 strain was the least antigenic of the set, in disagreement with previously reported studies with the virus isolate. Increasing peptide size from 15 to 21 residues did not significantly improve antigenicity. Overnight incubation of A15S30 with mAb 4C4 in solution showed a marked increase in peptide antigenicity not observed for other peptide analogues, suggesting that conformational rearrangement could lead to a stable peptide-antibody complex. In fact, peptide cyclization clearly improved antigenicity, confirming an antigenic reversion in a multiply substituted peptide. Solution NMR studies of both linear and cyclic versions of the antigenic loop of FMDV C-S30 showed that structural features previously correlated with antigenicity were more pronounced in the cyclic peptide.Twenty-six synthetic peptides, corresponding to all possible combinations of five single-point antigenicity-enhancing replacements in the GH loop of FMDV C-S8c1, were also studied. SPR kinetic screening of these peptides was not possible due to problems mainly related to the high mAb affinities displayed by these synthetic antigens. Solution affinity SPR analysis was employed and affinities displayed were generally comparable to or even higher than those corresponding to the C-S8c1 reference peptide A15. The NMR characterisation of one of these multiple mutants in solution showed that it had a conformational behaviour quite similar to that of the native sequence A15 and the X-ray diffraction crystallographic analysis of the peptide - mAb 4C4 complex showed paratope - epitope interactions identical to all FMDV peptide - mAb complexes studied so far. Key residues for these interactions are those directly involved in epitope - paratope contacts (141Arg, 143Asp, 146His) as well as residues able to stabilise a particular peptide global folding. A quasi-cyclic conformation is held up by a hydrophobic cavity defined by residues 138, 144 and 147 and by other key intrapeptide hydrogen bonds, delineating an open turn at positions 141, 142 and 143 (corresponding to the Arg-Gly-Asp motif). / Se diseñó un método rápido y sencillo para el análisis cinético por resonancia de plasmón superficial (RPS) de las interacciones entre antígenos peptídicos de bajo peso molecular (< 1600 Da) y anticuerpos monoclonales (AM) inmovilizados en la superficie de un chip sensor. Dichas interacciones se ajustaron a un modelo de interacción bimolecular 1:1 y las constantes cinéticas obtenidas resultaron fiables y reproducibles. Los parámetros clave para la calidad de las constantes cinéticas medidas fueron un flujo de tampón elevado, una densidad superficial de AM intermedia y una elevada concentración de péptido. El método se extendió a más de 40 análogos peptídicos frente a dos AM contra el virus de la fiebre aftosa (VFA), obteniéndose total correlación con datos anteriores de ELISA competitivo.Se sintetizaron once pentadecapéptidos con todas las combinaciones posibles de las cuatro mutaciones que caracterizan el bucle GH del aislado C-S30 del VFA respecto a la secuencia de referencia C-S8c1. Los resultados del análisis cinético directo, por RPS, de la antigenicidad de estos péptidos frente a tres AM sugirieron que dichas combinaciones eran aditivas, observación que fué confirmada por ELISA competitivo. Así, el tetramutante (A15S30) que mimetiza el bucle GH de C-S30 resultó ser el peor antígeno de la serie, en contraste con resultados anteriores con este aislado. Aumentando el tamaño del tetramutante de 15 a 21 aminoácidos no afectó significativamente su antigenicidad. En cambio, una incubación prolongada con el AM llevó a un aumento de reactividad no observado para otros análogos. Posiblemente una reordenación conformacional del péptido pudo conllevar a la formación de un complejo estable con el anticuerpo. Experimentos de RPS con un análogo cíclico del péptido A15S30 confirmaron una reversión en la antigenicidad del tetramutante inducible a través de restricciones conformacionales. Estudios de ambos péptidos, lineal y cíclico, por resonancia magnética nuclear (RMN) mostraron que características estructurales anteriormente correlacionadas con la antigenicidad eran más pronunciadas en el análogo cíclico.Se prepararon veintiseis péptidos con todas las posibles combinaciones de cinco sustituciones específicas en el bucle GH del VFA C-S8c1. Dichas sustituciones individuales habían sido objeto de estudios anteriores, obteniéndose una elevada antigenicidad para los correspondientes péptidos mutantes frente a AM anti-VFA. No se pudo sistematizar el análisis cinético por RPS de los nuevos mutantes multiples, debido a problemas tanto en la determinación de las constantes cinéticas de disociación, como en la regeneración de las superficies de AM. Se utilizó así la RPS para la determinación de la afinidad péptido - AM en solución, obteniéndose antigenicidades comparables o incluso superiores a las del péptido nativo A15 (VFA C-S8c1). Se estudió uno de los mutantes multiples (A15FPS) por RMN, observándose una conformación identica a la del péptido nativo. El estudio del complejo cristalino entre el péptido A15FPS y el AM 4C4 por difracción de RX mostró que las interacciones parátopo - epítopo eran similares a las observadas con el péptido nativo. Se concluyió que los residuos clave para el reconocimiento son tanto aquellos involucrados en contactos directos (141Arg, 143Asp, 146His) como aquellos que estabilizan el plegamiento adecuado del péptido. Así, una conformación casi cíclica es soportada por una cavidad hidrofóbica definida por los residuos 138, 144 y 147 y por puentes de hidrógeno intra-peptídicos clave, diseñándose un bucle abierto centrado en las posiciones 141, 142 and 143 (triplete Arg-Gly-Asp).

Interactions between carbohydrates and proteins are increasingly being recognized as crucial in many biological processes, such as cellular adhesion and communication. In order to investigate the interactions of carbohydrates and proteins, the development of efficient analytic technologies, as well as novel strategies for the synthesis of carbohydrates, have to be explored. To date, several methods have been exploited to analyze interactions of carbohydrates and proteins, for example, biosensors, nuclear magnetic resonance (NMR); enzyme-linked immunosorbent assays (ELISA), X-ray crystallography and array technologies. This thesis describes the development of novel strategies for the synthesis of carbohydrates, as well as new efficient strategies to Quartz Crystal Microbalance- (QCM-) biosensors and carbohydrate microarrays technologies. These methodologies have been used to probe carbohydrate-lectin-interactions for a range of plant and animal lectins. / QC 20100915

The detection and quantification of biomolecules, and proteins in particular, are of great interest since these molecules are of fundamental importance to our well-being. Body fluids, as for instance human blood, are well suited for sampling of protein levels. However, the complexity of the fluids and the low abundance of many of the interesting biomolecules makes detection and quantification difficult. This has spurred an interest into the development of many protein detection methods, and of these, ligand assays have proven particularly suitable. In this thesis, different types of ligand assays for protein detection have been developed using affibody molecules as ligands. In a first study, a homogeneous competitive detection assay was investigated, based on antiidiotypic affibody molecule pairs and fluorescence resonance energy transfer (FRET) as reporting system. The individual members of two anti-idiotypic affibody pairs, each consisting of a target binding (idiotypic) and an anti-idiotypic affibody ligand, were labeled with a donor fluorophore and an acceptor fluorophore, respectively. Incubation with the two target proteins IgA and Taq DNA polymerase resulted in a concentration dependent decrease in the FRET signal, allowing for target protein detection and quantification. For Taq DNA polymerase, detection in 25% human plasma was also possible in the same concentration span as in buffer. In a second study, a homogeneous, non-competitive detection system was described. Affibody molecules of 58 amino acids directed against IgA and IgG were produced with chemical synthesis, and two fluorophores capable of FRET were site-specifically introduced. Binding of target protein induced a concentration-dependent change in the relative emission of the two fluorophores, which formed the basis for the detection system. In two studies, affibody molecules were evaluated and shown to function well as capture ligands on microarrays. Synthetic affibody molecules directed against Taq DNA polymerase and IgA were modified by the introduction of immobilization tags. Specific immobilization via a C-terminal cysteine or a biotin moiety, or random immobilization via amino groups, were studied in protein microarray experiments and SPR-based biosensor studies. The experiments showed that all immobilization chemistries resulted in functional capture molecules. A short spacer was also introduced, situated between the affibody and the cysteine and biotin moieties, which was shown to improve binding for all constructs. Multidomain affibody constructs of up to four N- to C-terminally linked domains were shown to increase the amount of bound target, compared to monomeric affibody ligands. Six dimeric affibody constructs directed against IgA, IgG, IgE, Taq DNA polymerase, TNF-α and insulin, respectively, showed low limits of detections for their targets and little or no cross-reactivity with the other target proteins. Dimeric affibody molecules directed against IgA and TNF-α were also shown to function in a sandwich format with antibodies for detection of targets in buffer and in human serum and plasma. Successful discrimination between normal and IgA-deficient sera showed that affibody molecules could be used for specific detection of protein in highly complex backgrounds on microarrays. / QC 20100916

There is a great demand for low-cost disposable sensors in a variety of markets, such as the food chainand health care. No assay is performed more than that of glucose and approximately 85 % of the entirebiosensor market accounts for glucose biosensors. Each year, 6 billion glucose assays are performed andthe majority of them are based on electrochemical detection. Organic electrochemical transistors(OECTs) have favorable properties in terms of low operating voltages and have previously been used asbase for electrochemical detection of glucose. A low-cost disposable biosensor can be achieved by theuse of high throughput printing techniques. Up until now, no printable biosensors based on organic electrochemicaltransistors have been developed. In this thesis a printable miniaturized prototype for a glucose biosensor based on an OECT with a platinizedgate electrode has been designed, developed and evaluated. The biosensor has been functionalizedwith the enzyme glucose oxidase. Different platinum deposition techniques have been used to depositplatinum onto the printed carbon gate electrode: electrodeposition, platinum nanoparticle solutiondeposited either by inkjet printing or pipetting and thermal evaporation. The gate electrodes were characterized with cyclic voltammetry in hydrogen peroxide, ferricyanide andglucose. The characterizations revealed no significant differences between the different deposition techniques.However, with gate electrodes produced by printed carbon followed by electrodeposition ofplatinum it was possible to sense glucose in a concentration in the range of the values for diabetic persons.Thus, the electrodes are a promising option as gate electrodes in a glucose biosensor based on anOECT. The characteristics of the OECT revealed that the responses resembled a transistor.