With the development of ultra-fast laser technology, several new imaging techniques have pushed optical resolution past the diffraction limit for traditional light-based optics. Advancements in lithography have enabled the straightforward creation of micron- and nanometer-sized optical devices. Exposing metal-dielectric structures to light can result in surface plasmon excitation and propagation along the transition interface, creating a surface plasmon polariton (SPP) response. Varying the materials or geometry of the structures, the plasmonic response can be tailored for a wide range of applications. Photoemission electron microscopy (PEEM) has been used to image excitations in micron-sized plasmonic devices. With PEEM, optical responses can be characterized in detail, aiding in the development of new types of plasmonic structures and their applications. We show here that in thin, triangular gold platelets SPPs can be excited and concentrated within specific regions of the material (thickness ~50 nm); resulting in localized photoemission in areas of high electric field intensity. In this regard, the platelets behave as receiver antennas by converting the incident light into localized excitations in specific regions of the gold platelets. The excited areas can be significantly smaller than the wavelength of the incident light (λ≤1µ). By varying the wavelength of the light, the brightness of the excited spots can be changed and by varying the polarization of the light, the brightness and position can be changed, effectively switching the photoemission on or off for a specific region within the triangular gold structure. In this work, the spatial distribution of surface plasmons and the imaging results from photoemission electron microscopy are reproduced in simulation using finite element analysis (FEA). In addition, we show that electromagnetic theory and simulation enable a detailed and quantitative analysis of the excited SPP modes, an explanation of the overall optical responses seen in PEEM images, and prediction of new results.

The purpose of this study was to prepare concentrated and stable gold colloids for the enhancement of the signal response of the SPR technique for detecting small molecules such as progesterone. The gold colloids developed in this study were prepared by hydrazine hydrate, sodium borohydride, and tri-potassium citrate reduction routes. The study revealed that the sodium borohydride reduced gold colloids were extremely stable and it was able to be utilised in the progesterone immunoassay developed previously by Mitchell et al. The experiment was carried out on BIAcore 3000 using two different sensor surfaces (CM5 and SAM). The results showed that the enhancement species prepared from the borohydride-reduced gold colloids were able to improve the SPR signal response by 13 times higher than SPR signal produced without the enhancement species on the CM5 surface. The signal enhancement on the SAM surface using the same enhancement species was even greater at 29 times higher. The sensitivity of the assay was, however, unable to be determined due to time constraint. The limit of detection (LOD) of the progesterone assay using the CM5 chip was estimated to be ca. 5-20 pg/mL. Whilst for the SAM chip, the LOD of the progesterone assay was estimated to be ca. 5-20 fg/mL. Further work is required to confirm these estimated LOD values.

The interactions of biomolecules and cells at solid-liquid interfaces play a pivotal role in a range of biomedical applications and have hence been studied in detail. An improved understanding of these interactions results in the ability to manipulate biomolecules and concurrently cells spatially and temporally at surfaces with high precision. Spatial control can be achieved using patterned surface chemistries whilst temporal control is achieved by switchable surfaces. The combination of these two surface properties offers unprecedented control over the behaviour of biomolecules and cells at the solid-liquid interface. This is particularly relevant for cell microarray applications, where a range of biological processes must be duly controlled in order to maximise the efficiency and throughput of these devices. Of particular interest are transfected cell microarrays (TCMs), which significantly widen the scope of microarray genomic analysis by enabling the high-throughput analysis of gene function within living cells Initially, this thesis focuses on the spatially controlled, electro-stimulated adsorption and desorption of DNA. Surface modification of a silicon chip with an allylamine plasma polymer (ALAPP) layer resulted in a surface that supported DNA adsorption and sustained cell attachment. Subsequent high density grafting of poly(ethylene glycol) (PEG) formed a layer resistant to biomolecule adsorption and cell attachment. PEG grafted surfaces also showed significantly reduced attachment of DNA with an equilibrium binding constant of 23 ml/mg as compared with 1600 ml/mg for ALAPP modified surfaces. Moreover, both hydrophobic and electrostatic interactions were shown to contribute to the binding of DNA to ALAPP. Spatial control over the surface chemistry was achieved using excimer laser ablation of the PEG coating which enabled the production of patterns of re-exposed ALAPP with high resolution. Preferential electro-stimulated adsorption of DNA to the ALAPP regions and subsequent desorption by the application of a negative bias was observed. Furthermore, this approach was investigated for TCM applications. Cell culture experiments demonstrated efficient and controlled transfection of cells. Electro-stimulated desorption of DNA was shown to yield enhanced solid phase transfection efficiencies with values of up to 30%. The ability to spatially control DNA adsorption combined with the ability to control the binding and release of DNA by application of a controlled voltage enables an advanced level of control over DNA bioactivity on solid substrates and lends itself to biochip applications. As an alternative approach to surface patterning, the fabrication and characterisation of chemical patterns using a technique that can be readily integrated with methods currently used for the formation of microarrays is also presented. Here, phenylazide modified polymers were printed onto low fouling ALAPP-PEG modified surfaces. UV irradiation of these polymer arrays resulted in the crosslinking of the polymer spots and their covalent attachment to the surface. Cell attachment was shown to follow the patterned surface chemistry. Due to the use of a microarray contact printer it was easily possible to deposit DNA on top of the polymer microarray spots. A transfected cell microarray was generated in this way, demonstrating the ability to limit cell attachment to specific regions and the suitability of this approach for high density cell assays. In order to allow for the high-throughput characterisation of the resultant polymer microarrays, surface plasmon resonance imaging was utilised to study the adsorption and desorption of bovine serum albumin, collagen and fibronectin. This analysis enabled insights into the underlying mechanisms of cell attachment to the polymers studied. For the system analysed here, electrostatic interactions were shown to dominate cellular behaviour.

Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilisation of a protein in the reference channel gives a better shielding effect between the negatively charged heparin molecules and the negatively charged dextran matrix. Furthermore a more significant difference was seen between the two heparin moieties used during binding affinity studies, especially for native AT.

Structure-specific recognition of nucleic acids is a promising method to reduce the size of the recognition unit required to achieve the necessary selectivity and binding affinity for small molecules. It has been demonstrated recently that G-quadruplex DNA structures can be targeted by organic cations in a structure-specific manner. Structural targets of quadruplexes include the planar end surfaces of the G-tetrad stacked columns as well as four grooves. The significant structural differences between quadruplex DNA and duplex DNA make quadruplex DNA a very attractive target for highly selective, structure-specific drug design. We have used a variety of biophysical techniques including circular dichroism, surface plasmon resonance, thermal melting and absorbance spectroscopy to investigate small molecules that can selectively bind to the ends of human telomeric DNA as well as the ends of the G-quadruplex structure formed by the purine-rich promoter region of the c-MYC oncogene. We have also screened a library of heterocyclic diamidines, and identified one that binds selectively in the grooves of human telomeric quadruplex DNA. This compound is an excellent starting point for the design of new anti-cancer and anti-parasitic compounds with high affinity and selectivity for human telomeric DNA.

In this project a method based on surface plasmon resonance has been developed for determining the concentration of several His-tagged proteins in complex solutions. It showed large dynamic range, no measureable non-specific binding and high sensitivity (with linear range around 0.1–10 μg/ml depending on the proteins). The method showed a low variation when checked on MBP-His during an extended time period. The concentrations of the His-tagged protein in the lysate has also been determined and compared with other alternative methods. This method will later be used to analyse protein concentrations during development and optimization of chromatographic purification process.

Electron-excited surface plasmon resonance (eSPR) is investigated for potential use in biosensors. Optical SPR sensors are commercially available at present and these sensors are extremely sensitive, but have the tendency to be relatively large, expensive, and ignore the potentials of microelectronic technology. By employing the use of various microelectronic and nanotechnology principles, the goal is to eventually design a device that exploits the eSPR phenomenon in order to make a sensor which is siginificantly smaller in size, more robust, and cheaper in cost.

<p>Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilisation of a protein in the reference channel gives a better shielding effect between the negatively charged heparin molecules and the negatively charged dextran matrix. Furthermore a more significant difference was seen between the two heparin moieties used during binding affinity studies, especially for native AT.</p>

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 198-210).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 164-178).