Food poisoning is mainly caused by pathogenic microorganisms and is now a severe problem worldwide. Therefore, rapid and sensitive methods are required to detect foodborne pathogens. A locally isolated bacterium, Bacillus licheniformis B38 was selected for this study. The spores of B. licheniformis B38 were induced by Schaeffer’s sporulation medium containing KCl, MgSO4.7H2O, Ca(NO3)4, MnCl2 and FeSO4. Schaeffer-Fulton endospore staining was used to differentiate spores and vegetative cells, where spores were stained green and vegetative cells were stained red. In order to separate the spores from the cells, a two-phase system was used to obtain pure spore suspension for following experiments. Spore coat proteins were extracted by SDS-8 M urea sample buffer and visualized by two different types of coomassie brilliant blue staining solutions. One of the staining solutions was more suitable for gel elution by diffusion. An ~10 kDa spore coat protein was selected for protein purification. Based on the given results, the protein purification by liquid chromatography was less convincing than using gel elution by diffusion technique. The two hypothetical protein sequences, P06552 and P45693, from the ~10 kDa spore coat protein were identified. In the preliminary study of B. licheniformis B38 spores detection by surface plasmon resonance, several binding parameters were studied. Dot blot was done to verify the reaction between the Bacillus spores polyclonal antibody against the B. licheniformis B38 spore coat protein. The most promising result was the binding of 0.1 mg/mL polyclonal antibody (analyte) to the 0.2 mg/mL spore coat protein at pH 2 (ligand) which showed 5.74 RU. The differences between a dot blot and a SPR detection techniques are described.

Cancer has become one of the leading causes of death today. The early detection of cancer may lead to desired therapeutic management of cancer and to decrease the mortality rate through effective therapeutic strategies. Advances in materials science have enabled the use of nanoparticles for added contrast in various imaging techniques. More recently there has been much interest in the use of gold nanoparticles as optical contrast agents because of their strong absorption and scattering properties at visible and near-infrared wavelengths. Highly proliferative cancer cells overexpress molecular markers such as epidermal growth factor receptor (EGFR). When specifically targeted gold nanoparticles bind to EGFR they tend to cluster thus leading to an optical red-shift of the plasmon resonances and an increase in absorption in the red region. These changes in optical properties provide the foundation for photoacoustic imaging technique to differentiate cancer cells from surrounding benign cells. In photoacoustic imaging, contrast mechanism is based on the optical absorption properties of the tissue constituents. Studies were performed on tissue phantoms, ex-vivo and in-vivo tumor models to evaluate molecular specific photoacoustic imaging technique. The results indicate that highly sensitive and selective detection of cancer cells can be achieved by utilizing the plasmon resonance coupling effect of EGFR targeted gold nanoparticles and photoacoustic imaging. In conclusion, the combined ultrasound and photoacoustic imaging technique has the ability to image molecular signature of cancer using bioconjugated gold nanoparticles. / text

Thermophotovoltaic (TPV) devices, also known as (nano-TPVs) are energy-conversion systems which generate electric current from thermal radiation energy by a heat source. Although their conversion efficiency is limited in the far field by the Schockley-Queisser limit, in near field the heat flux transferred to a TPV cell can be significantly enchanced due to the contribution of evanescent waves, in particular supporting a surface mode. Unfortunately, spectral mismatch between the emitter and the TPV cell spectrum limits the TPV conversion efficiency. Photons with energy lower than the TPV cell bandgap may not be able to create electron-hole pairs because mobile carriers start diffusing and drifting between conductance and valence band, and try to exceed the upper limit of the band. This destroys the thermal equilibrium of the semiconductor and results in excess heat. Also, for high energy photons, the difference between the photon's energy and the bandgap energy is lost in Joule heating. Thus, quasimonochromatic, narrow-band and coherent emitters at a frequency near the energy bandgap of the converter is an ideal source to achieve high conversion efficiency. Nano-TPV device consisting of tungsten thermal emitter, maintained at 1200K, and the cell made of GaInAsSb are considered; thermal management system is reviewed assuming a constant heat flux boundary due to heat generation by the cell with a fluid temperature fixed at 293K. Tungsten thermal selective emitters are designed, characterized and optimized based on two-dimensional (2D) tungsten PhC by controlling periodic triangular grooves such that channel plasmon polaritons (CPPs) are coupled efficiently into these grooves to excite a localized groove modes which are well-matched to the GaInAsSb cell external quantum efficiency (EQE). The results show that power output and the 2D TE normal efficiency of the system are predicted to be 0.82x10⁴ W/m² and 43.8%, respectively. This leads to a promising device for many different sectors such as military, space and semiconductor industry. / text

Optical whispering-gallery mode (WGM) cavities, which exhibit extraordinary spatial and temporal confinement of light, are one of the leading transducers for examining molecular recognition at low particle counts. With the advent of hybrid photonic-plasmonic and increasingly sophisticated forms of these resonators, the importance of supporting numerical methods has correspondingly become evident. In response, we adopt a full-vector finite difference approximation in order to solve for WGM's in terms of their field distributions, resonant wavelengths, and quality factors in the context of naturally discontinuous permittivity structure. A segmented Taylor series and alignment/rotation operator are utilized at such singularities in conjunction with arbitrarily spaced grid points. Simulations for microtoroids, with and without dielectric nanobeads, and plasmonic microdisks are demonstrated for short computation times and shown to be in agreement with data in the literature. Constricted surface plasmon polariton (SPP) WGM's are also featured within this document. The module of this thesis is devised as a keystone for composite WGM models that may guide experiments in the field. / Graduate

Small molecules interacting with DNA is an emerging theme in scientific research due to its specificity and minimal side-effect. Moreover, a large amount of research has been done on finding compounds that can stabilize G-quadruplex DNA, a non-canonical secondary DNA structure, to inhibit cancerous cell proliferation. G-quadruplex DNA is found in the guanine-rich region of the chromosome that has an important role in protecting chromosomes from unwinding, participate in gene expression, contribute in the control replication of cells and more. In this research, rationally designed, synthetic cyanine dye derivatives, which were tested under physiologically relevant conditions, were found to selectively bind to G-quadruplex over duplex DNA and are favored to one structure over another. The interactions were observed using UV-Vis thermal melting, fluorescence titration, circular dichroism titration, and surface plasmon resonance analysis. For fluorescence and selectivity properties, cyanine dyes, therefore, have the potential to become the detections and/or therapeutic drugs to target cancers and many other fatal diseases.

Optical manipulation of nanoscale objects is studied with particular emphasis on the role of plasmon resonance for enhancement of optical forces. The thesis provides an introduction to plasmon resonance and its role in confinement of light to a sub-diffraction volume. The strong light confinement and related enhancement of optical forces is then theoretically studied for a special case of nanoantenna supporting plasmon resonances. The calculation of optical forces, based on the Maxwell stress tensor approach, reveals relatively weak optical forces for incident powers that are used in typical realisations of trapping with nanoantenna. The optical forces are so weak that other non-optical effects should be considered to explain the observed trapping. These effects include heating induced convection, thermoporesis and chemical binding. The thesis also studies the optical effects of plasmon resonances for a fundamentally different application - size-based optical sorting of gold nanoparticles. Here, the plasmon resonances are not utilised for sub-diffraction light confinement but rather for their ability to increase the apparent cross-section of the particles for their respective resonant sizes. Exploiting these resonances, we realise sorting in a system of two counter-propagating evanescent waves, each at different wavelength that selectively guide gold nanoparticles of different sizes in opposite directions. The method is experimentally demonstrated for bidirectional sorting of gold nanoparticles of either 150 or 130 nm in diameter from those of 100 nm in diameter within a mixture. We conclude the thesis with a numerical study of the optimal beam-shape for optical sorting applications. The developed theoretical framework, based on the force optical eigenmode method, is able to find an illumination of the back-focal plane of the objective such that the force difference between nanoparticles of various sizes in the sample plane is maximised.

Thrombospondin-1 (TSP-1) binding to its membrane receptor CD47 results in an inhibtion of the nitric oxide (NO) receptor soluble guanylate cyclase (sGC) and a decrease in intracellular cGMP levels. This causes physiologic effects such as vasoconstriction and a rise in blood pressure. The mechanism by which TSP-1 binds to CD47 at the membrane to decrease sGC activity is largely unknown. CD47 can physically associate with a number of binding partners, including α(v)β₃ and vascular endothelial growth factor receptor 2 (VEGFR2). Binding of a C-terminal fragment of TSP-1 called E3CaG1 to CD47 leads to a rise in intracellular calcium ([Ca²⁺](i)), which decreases sGC activity via a phosphorylation event. Binding of E3CaG1 is also known to disrupt the interaction between CD47 and VEGFR2, leading to a decrease in endothelial nitric-oxide synthase (eNOS) activity and cGMP levels through an Akt signaling pathway. However, it is not known whether other membrane proteins associated with CD47 are required for E3CaG1 binding and a subsequent [Ca²⁺](i) increase. Plasmon-waveguide resonance (PWR) spectroscopy was employed to elucidate the mechanism of TSP-1 inhibition of sGC activity through membrane complexes involving CD47. Using PWR, I found E3CaG1 can bind specifically to CD47 within native Jurkat membranes with picomolar and nanomolar dissociation constants (K(d)), suggesting multiple CD47 complexes are present. Among these complexes, CD47/VEGFR2 was found to bind E3CaG1 with a picomolar K(d)and CD47/α(v)β₃ was found to bind E3CaG1 with a nanomolar K(d). In addition, the presence of an anti-VEGFR2 antibody inhibited the E3CaG1-induced calcium response, which suggested CD47 in complex with VEGFR2 was responsible for TSP-1 reduction of sGC activity. I show that when both CD47 and VEGFR2 are returned to a HEK 293T cell line that does not contain these receptors, an increase in [Ca²⁺](i) upon E3CaG1 binding is restored. Interestingly, E3CaG1 was also found to bind to VEGFR2 in complex with the integrin α(v)β₃ on CD47-null cell lines and their derivations, causing a decrease in [Ca²⁺](i) levels. Therefore, the third type 2 repeat and C-terminal domains of TSP-1 can cause both increases and decreases in calcium based upon the availability of protein complexes to which it binds.

The tumor microenvironment is a tangled web of multiple cell types, extracellular matrix components, and a multitude of cell signaling pathways frequently contribute to poor outcomes, which make cancer the second leading killer in the United States. A better understanding of how these constituents interact will inevitably facilitate development of novel cancer therapeutics and diagnostics. To advance scientific discovery towards this goal, innovative experimental techniques are required. In this dissertation, new research methods for probing cell communication at a single to multi cell level within 3D models of the tumor microenvironment are presented. Optical trapping, composite nanocapsules (i.e., gold-coated liposomes), and 3D cell culture models were the foundation for the development of these research tools. The first aim of this dissertation was to optimize our ability to optically manipulate gold-coated liposomes for the purpose of delivering molecular content to cells. The second aim was to apply optical manipulation of gold-coated liposomes to quantitatively deliver signaling molecules into a single cell to activate communication. The third aim was to develop a 3D model of the tumor microenvironment and demonstrate cell communication within this physiologically accurate architecture. The basis for this work was gold-coated liposomes' strong plasmon resonance with visible to near infrared (NIR) wavelengths of light, which enabled photo-thermal conversion and optical trapping. To identify preferred conditions for optical manipulation of gold-coated liposomes for delivering content into cells, gold-coated liposomes made with different dielectric properties were optically trapped under various laser modulation schemes and thoroughly characterized, enabled by high speed (kHz) imaging. Application of this technique was realized by precise delivery of molecular agents into a single cell (i.e., optical injection). As a demonstration of optical injection, the NIR trapping beam was utilized to propel gold-coated liposomes encapsulating inositol trisphosphate (IP3) into a single cell to initiate calcium (Ca²⁺) signaling. In another method for intracellular delivery, cells were preloaded with similar gold-coated liposomes, internalized by macropinocytosis, and then exposed to on-resonant laser light to trigger on-demand release of IP3 to activate Ca²⁺ signaling. Lastly, a 3D cell culture model of ovarian cancer microenvironment was developed as a platform for interrogating cell signaling. The in vitro model comprised human ovarian cancerous epithelial cells grown upon a collagen and human fibroblast stroma recapitulating architecture of human tissue. Gold-coated liposomes encapsulating signaling molecules, optical manipulation, and a 3D model of ovarian cancer, a trio of versatile experimental tools opens new opportunities for studying the tumor microenvironment.

Advanced optical imaging techniques are emerging as useful ways to screen tissues for the presence of cancer. Plasmon resonant nanoparticles have unique optical properties that make them ideal for use as optical contrast agents. The capacity of these particles to serve a multifunctional role dependent on their composition and the intensity of incident light enables them to serve as diagnostic tools and to provide the therapeutic capability of photo-thermal energy conversion or the controlled release of an encapsulated agent. Likewise, the ability to degrade into components of a clearable size may enable the clinical translation of these types of particles.These properties were demonstrated by means of experiments in the support of three specific aims. The first specific aim was to determine whether the unique and tunable optical properties of nanorods lend them to generate signal in advanced optical imaging techniques, and that nanorods can facilitate photo-thermal conversion. The second specific aim was to show that liposomes can serve as a scaffold for the support of an array of gold nanodots to generate a structure that exhibit tunable plasmon resonant characteristics and a resultant ability to generate signal in optical imaging techniques while having the capability to degrade into inert particles of a size that can be readily cleared from the body via the kidney. The final specific aim was to determine whether the gold-coated liposomes of the second specific aim can serve as system for light-based delivery of an encapsulated agent in addition to its role as an optical contrast agent and its biodegradation capacity.Plasmon resonant nanorods and plasmon resonant gold-coated liposomes were generated by reducing free gold from solution onto surfactant coated seed particles and phospholipid liposomes, respectively. Both structures demonstrated the ability to generate signal in optical coherence tomography and in multi-photon confocal microscopy images. Nanorods in high intensity light demonstrate a capacity to mediate photo-thermal energy conversion. While, in similar conditions, gold-coated liposomes are shown to release their contents. Gold-coated liposomes are also shown to degrade to bioinert components of a size reasonable for rapid renal clearance using either surfactant or enzyme.

With the manufacture of nano-scale features in the last ten years, it is possible to do optical experiments on features as small as a tenth/hundredth wavelength. It turns out that the experimental data cannot be explained by classical diffraction theories. Thus, it is necessary to develop new methods or use existing approaches which are effective in other fields, to solve problems in photonics. We use finite difference time domain (FDTD), to study transmission properties of sub-wavelength slits in a metallic film. By doing simulations on periodic and single slits, we confirm that the TE mode has a cutoff while a TM mode always has a propagating mode in the small apertures. Then we find that the transmittance is minimum when the array period is equal to the wavelength of surface plasmon polariton (SPP) at normal incidence. In fact, the SPP-like waves exist in both periodic and isolated slits, and they help the transmittance of small apertures. In order to establish the role of SPP in the transmission mechanism, it is necessary to single out each mode from the total fields. We developed Bloch mode method (BMM) to calculate the amplitudes of the lowest N orders, and the amplitudes tell us which one is dominant (not including the guided mode) at high and low transmission. BMM converges very fast and it is more accurate than FDTD since it does not suffer from numerical dispersion. Both methods can resolve the Wood anomaly and SPP anomaly; however, FDTD converges very slowly at the SPP resonance and oscillates around the value obtained through BMM at the Wood anomaly. BMM is not sensitive to material types, incident angles, and anomalies; it will be a useful tool to investigate similar problems.