The aims of this study are to first immobilize positively charged gold nanorods to negatively charged cell culture surfaces. Second, to use polyelectrolytes for controlling the distance between gold nanorods and fluorophores. This is used to optimally determine the distance, of which maximum fluorescence enhancement is achieved, between gold nanorods and fluorophores. In order to approach these aims, we use UV/VIS absorption spectroscopy, fluorescence spectroscopy, atomic force microscopy, and ellipsometry. The results show that we could control the immobilization of gold nanorods on plastic microwell plates and create reproducible polyelectrolyte layers, in order to control the distance between the gold nanorods and fluorophores. In addition, the localized surface plasmon resonance wavelength red shifted as the PELs increased. In conclusion, we found that the maximum fluorescence enhancement of the fluorophores (Cy7) is about 2.3 times at a fluorophores-nanoparticles separation of approximately 9-12 nm. This work contributes some research information towards the design of optical biochip platforms based on plasmon-enhanced fluorescence.

Recently, technological advancement and the promise of next-generation devices have created an overwhelming push for the continued miniaturization of active systems to the micro- and nanometer scale. In this regime, traditional mechanical systems are largely inaccessible and as a result new active or stimuli-responsive materials are required. The work presented in this dissertation provides an understanding of the responsive nature of polymer and biopolymer interfaces especially in contact with metal nanoparticles. This understanding was utilized in conjunction with top-down template-based and self-assembly fabrication strategies to create hybrid protein based films and active polymer-metal hybrids that exhibit large and well-defined modulation of mechanical and optical properties. These materials processing developments represent advancement in the current state of the art specifically in three major areas: 1. template-based top-down control of protein chain conformation, 2. high-throughput synthesis and assembly of strongly coupled plasmonic nanoparticles with modulated optical properties (both near- and far-field), 3. field-assisted assembly of highly mobile and non-close packed magnetic nanorods with capabilities for rapid actuation.

There is a high demand for quadruplex-specific compounds that not only bind preferentially to quadruplex DNA over duplex DNA, but also bind to one quadruplex motif over other motifs. Quadruplex structures are recognized as common occurrences in cancer cells, and if a compound could stabilize this structure, it may serve as an effective anti-cancer treatment with minimal side effects. In this study, cyanine dyes’ interactions with DNA were analyzed with fluorescence titrations, UV-Vis thermal studies, circular dichroism titrations, and surface plasmon resonance (SPR) analysis. With these techniques, binding affinity, DNA stabilization, and conformational shifts were analyzed to determine if cyanine dyes could act as quadruplex-specific binding compounds for possible cancer treatments.

The development of small-molecules which target nucleic acids, more specifically the minor groove of DNA, in a sequence specific manner and control gene expression are currently being investigated as potential therapeutic compounds for the treatment of various diseases, including cancer, as well as viral and bacterial infections. The naturally occurring compounds netropsin and distamycin have been shown to demonstrate antitumor and antibacterial properties. Currently, there are synthetic efforts to create pyrrole and imidazole-containing polyamide derivatives of netropsin and distamycin that show potential as medicinal agents. Synthetic pyrrole and imidazole-containing polyamides are potentially useful for targeting and modulating the expression of genes, including those associated with cancer cell growth. The key challenges that must be overcome to realize this goal of using synthetic polyamides in the treatment of disease are the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. As part of a systematic study within the authors’ laboratory, our goal is to develop polyamides which can be synthesized readily yet possess excellent sequence specificity, stronger binding affinity, high solubility in biological media and enhanced cell penetration and nuclear localization properties. There is a need to develop a library of modified polyamides which target DNA and exhibit improved biological properties. The present study is a systematic examination of the binding properties of various modified synthetic polyamide compounds. The synthetic polyamide derivatives presented have more potential as therapeutic candidates over other synthetic polyamides because of their increased water solubility, smaller molecular weights, and molecular design, thus, allowing them to penetrate into cells and localize in the nucleus.

Reliable analytical tools are important for time efficient and economical process development, production and batch release of pharmaceuticals. Therapeutics recovered from human plasma, called plasma protein products, involve a large pharmaceutical industry of plasma fractionation. In plasma fractionation of human immunoglobulin G (hIgG) and albumin (HSA) recommended analysis techniques are regulated by the European Pharmacopoeia and are including total protein concentration assays and zone electrophoresis for protein composition and purity. These techniques are robust, but more efficient techniques with higher resolution, specificity and less hands-on time are available. Surface plasmon resonance is an optical method to study biomolecular interactions label-free in real time. This technology was used in this master thesis to set up assays using Biacore systems for quantification of HSA and hIgG from all steps of chromatographic plasma fractionation as a tool for process development and in-process control. The analyses have simplified mass balance calculations to a high extent as they imply specific detection of the proteins compared with using total protein detection. The assays have a low hands-on time and are very simple to perform and the use of one master calibration curve during a full week decreases analysis time to a minimum. Quick, in-process control quantification of one sample is easily obtained within <10 minutes. For final QC of hIgG or for process development, an assay to quantify the distribution of the IgG subclasses (1-4) was set up on Biacore and showed significantly lower hands-on time compared with a commercial ELISA. All assays showed reliable quantification and identification performed in unattended runs with high precision, accuracy and sensitivity.

Biodegradable, edible wheat gluten films offer a renewable alternative to plastic food packaging or can be incorporated directly in the food product. Wheat gluten is a good option because it forms a fibrous network, lending strength and elasticity to films. The goal of this research project was to produce, with a water-based film formulation and methodology, smooth, homogeneous wheat gluten films with low water vapour permeability (WVP). The water-based film formulation also served to compare the FT Wonder wheat cultivar, grown in Ontario, to commercially produced wheat gluten and determine the effect of wheat source on the film properties, surface morphology, surface hydrophobicity, WVP, and film swelling in water for different pH, temperature and casting surface conditions. Fluorescence, SPR, and casting formulation viscosity provided preliminary information on the mechanism of film formation and on gluten protein structure induced by modifying the film formulation. This research provides an alternate use for some Ontario wheat cultivars based on their properties in films compared to commercial sources of gluten. As a result, using Ontario cultivars to prepare gluten film packaging material has potential as an alternate source of income for Ontario farmers. This research also defines the film properties for gluten films produced from aqueous solutions, helping to identify processing parameters that could bring gluten films on par with plastic packaging and make gluten films a viable alternative food packaging material. Finally, it was determined that the water vapour permeability of wheat gluten films was not correlated to film surface contact angle.

Surface plasmon waves are transverse magnetic electromagnetic waves propagating along a dielectric-metal interface. These waves can be excited by resonant absorption of electromagnetic radiation leading to surface plasmon resonance (SPR) at the interface. The resonance is characterised by a reduction in the intensity of the reflected light at the interface due to strong coupling of incident optical radiation to surface plasmons. This gives rise to a minimum at a sharply defined angle of incidence, referred to as SPR angle or plasmon angle. The phenomenon of SPR has been extensively used in the past to develop reflective type optical devices for sensing applications on account of the high dielectric function dependent sensitivity of the SPR angle. Basically, devices which exhibit this phenomenon have a structure consisting of a metal film sandwiched between two dielectrics. The reflectivity of such a device is theoretically modelled based on either theory of thin films (Fresnel's model) or theory of resonance (Lorentzian model). These models have very effectively predicted the behaviour of such devices based on the shift in SPR angle due to the dielectric function variations. We have been investigating the SPR device for intensity based metrological applications utilising its high angular sensitive reflectivity, with fixed SPR angle. In these intensity based applications or measurements, direct and simple expressions connecting intensity variation to angular change are unavailable in the literature and quantitative estimation or data inversion is based on either curve fitting or iterative methods. Fresnel and Lorentzian models have commonly been used in the experiments but data inversion through the Fresnel model is computationally complex and the Lorentzian model, although less complicated, gives erroneous results due to its approximate nature. In order to obtain a simple expression between intensity variation and the angular change, we have re-looked at the two existing models in order to derive an expression which has the simplicity of the Lorentzian model and the accuracy of the Fresnel model in the experiments with fixed plasmon angles. These efforts have been particularly directed to understand the relationship between intensity variation and meteorologically important properties of such devices. This thesis is an attempt to summarize the computational results which have led us to some novel experimental methodologies which have been used to exploit these devices for inverse type, illumination specific, SPR based applications. The work presented in this thesis is organised in six chapters. Chapter 1, gives an overview of optical sensing, theory of surface plasmons, excitation schemes for surface plasmons, development of the SPR device and its characterisation. It also includes a brief literature review in the area of surface plasmon resonance, covering both the theoretical and experimental aspects. The objectives of the work and the scope of the thesis are also presented. Chapter 2 presents the existing models of SPR device, based on Fresnel's and the Lorentzian models. These models allow reflectance calculations from knowledge of either the optical parameters that describe the layers or the parameters of the waves that propagate through them. Using these models, the inverse problem of estimating either the angle of incidence or the optical constants of the layers of the sensors utilizing the intensity based measurements is generally difficult. In order to solve this problem where the plasmon angles are fixed, a modified formalism for the angle scanned SPR spectrum of a three-layered SPR sensor is presented in this chapter. The new formalism regroups the wave vector parameters of Lorentzian resonance theory into a set of non-dimensional parameters 1, 4K and R. The new reflectivity index (1), which is the ratio of reflectance to the absorptance, has been introduced to help explain the physical processes underlying the device operation in the high sensitivity region of the characteristics. The parameter 4Kis a constant of the device and it depends on the dielectric constants of the device. This is a new SPR index and is identified at a point where reflectance and absorptance match. Parameter R is related to the loss mechanisms in the device and will be explained in detail in Chapter 3. This simple model links the new reflectivity index (1) to the angular detune from SPR angle (ΔƟ) and it brings out a parabolic variation of ΔƟ with 1. In this chapter the mathematical derivation of the proposed model is presented and the significance of the new parameters 1, 4Kand Rare discussed. Chapter 3 evaluates the characteristic nature of errors associated with the predictions from the proposed model and presents methods for neutralizing them. It is demonstrated with the help of the function K which is linearly dependant on 1, that the proposed model predicts the reflectance from the wave vector parameters as accurately as the Fresnel's model. This R parameter explains the slowly varying nature of the radiative loss with the angle of incidence and this variation contributes significantly to the SPR characteristics. As a consequence, it is found that the SPR characteristics can be represented as a sum of two primary functions which are parabolic and linear, respectively, and this leads to the easy explanation of the SPR characteristics. The present chapter also discusses a new observation that the angle-scanned SPR spectrum can be accurately described using a straight line in intercept form. The intercept value depends on 4Kand the slope depends on K. In addition to this, this chapter discusses practical methods for estimation of the intercept and the slope of such a straight line which are functions of the key wave vector parameters. A detailed discussion on the proposed model highlighting its advantages for inverse type, illumination specific, SPR-based applications with fixed SPR angle is also presented. Chapter 4 describes the applications of the proposed model for optical constant measurements. The first part highlights a new approach for the determination of the dielectric constants of the metal film used for the optimised- or nearly-optimised SPR sensors using the proposed model. In the complex dielectric constant, the real part is calculated from the SPR angle and the imaginary part from 4K. A discussion on the dielectric constant study of silver and gold metal film is presented. The advantages of the proposed approach such as its simplicity and direct methodology are then discussed. The second part of the chapter also proposes a new approach to carry out measurements on the absorbance of the medium with enhanced sensitivity utilising the parameter 4K It describes a computational study on the variation of 4K values with the dielectric function and highlights the relationship of 4K variation due to the imaginary part of the dielectric function (absorption) of the samples. The physical processes causing a change in the value of 4Kdue to absorption is also discussed along with some computational results. Chapter 5 reports the study carried out to bring out the importance of the new index,4K in metrological applications. Based on the new model, the effect of the laser beam divergence on SPR curve is studied. This chapter first of all discusses the design of the SPR device and the new methods for the development and characterisation of such a device. Details of the experimental procedure for laser divergence evaluation are proposed along with some of the significant computational results. Furthermore, a few applications such as focal length measurement of optical lenses, micro-displacement measurement based on the divergence of the laser beam are also reported. Since the SPR characteristics can be represented easily using the new model, the angular dependent intensity variation can be utilised for some metrological applications with simple data processing. In this context, the high angular sensitivity of the SPR device is studied and some applications such as micro-displacement measurement, pressure measurement and optical wedge angle measurement are included to highlight the above advantages. The last chapter, Chapter 6, gives a summary and conclusions of the work presented in the thesis. The scope for future investigations is also included in this chapter.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 177-181).

IgM monoclonal gammopathy is a common age-related demyelinating sensory and motor polyneuropathy. It has been shown to be associated with antibodies against myelin-associated glycoproteins (MAG/SGPG). The HNK-1 carbohydrate epitope is a terminal 3-sulfo-glucuronyl residue attached to lactosamine structures and it is shared both in MAG and SGPG (SO4-3-GlcA(β1-3)Gal-(β1-4)GlcNAc(β1-3)Gal-(β1-4)Glcβ(1-1′)Cer). It is mostly expressed in the nervous system and plays an important role in preferential motor reinnervation. Nevertheless, the HNK-1 epitope is difficult to be isolated and synthesized and diagnostic assays used in the clinics are not always reproducible and reliable. Therefore in our study, our goal is to identify a simple synthetic diagnostic tool (peptide or monosaccharide), mimetic of the HNK-1 epitope, able to recognize antibodies in neurogammopathies sera by Surface Plasmon Resonance to be used in earlier stage patients and possibly to monitor disease activity. For this reason, we firstly tried to synthesize this trisaccharide and then we achieved the synthesis of its terminal monosaccharides with different function groups (octyl glucopyranoside, octyl glucuronic acid, octyl 3-O-sulfo-glucuronic acid and 8-amino octyl 3-O-sulfo-glucuronic acid). Then 10 linear and cyclic peptides conformationally and/or structurally mimicking HNK-1 were also synthesized (LSETTI, LSETTl, cyclo(-TTILSE-), cyclo(-TTlLSE-), cyclo(-TKTlLSE-), cyclo(-TETKlLSE-), TYTKlLSE, TY(SO3)TKlLSE, cyclo(-TYTKlLSE-) and cyclo(-TY(SO3)TKlLSE-)). The SPR kinetic binding affinities of all these sugar and peptide mimics were studied with commercial anti HNK-1 antibody using Biacore. Moreover, mimics with highest binding affinities were chosen for antigen-antibody interaction study in IgM gammopathy patients' serum.

Noble metal nanoparticles supporting localized surface plasmon resonances (LSPR) have been extensively investigated for label free detection of various biological and chemical interactions. When compared to other optical sensing techniques, LSPR sensors offer label-free detection of biomolecular interactions in localized sensing volume solutions. However, these sensors also suffer from a major disadvantage – LSPR sensors remain highly susceptible to interference because they respond to both solution refractive index change and non-specific binding as well as specific binding of the target analyte. These interactions can severely compromise the measurement of the target analyte in a complex unknown media and hence limit the applicability and impact of the sensor. In spite of the extensive amount of work done in this field, there has been a clear absence of efforts to make LSPR sensors immune to interfering effects. The work presented in this document investigates, both experimentally and numerically, dual- and tri-mode LSPR sensors that utilize the multiple surface plasmon modes of gold nanostructures to distinguish target analyte from interfering bulk and non-specific binding effects. Finally, a series of biosensing experiments are performed to examine various regeneration assays for LSPR sensors built on indium tin oxide coated glass substrate.