Self-assembly of polysaccharide derivatives at liquid/solid interfaces was studied by surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Carboxymethyl cellulose (CMC) adsorption onto cellulose surfaces from aqueous solutions was enhanced by electrolytes, especially by divalent cations. A combination of SPR and QCM-D results showed that CMC formed highly hydrated layers on cellulose surfaces (90 to 95% water by mass). Voigt-based viscoelastic modeling of the QCM-D data was consistent with the existence of highly hydrated CMC layers with relatively low shear viscosities of ~ 10-3 N·s·m-2 and elastic shear moduli of ~ 105 N·m-2. Adsorption of pullulan 3-methoxycinnamates (P3MC) and pullulan 4-chlorocinnamates (P4CC) with different degrees of cinnamate substitution (DSCinn) onto cellulose, cellulose acetate propionate (CAP), poly(L-lactic acid) (PLLA), and methyl-terminated self-assembled monolayer (SAM-CH3) surfaces was also studied by SPR and QCM-D. Hydrophobic cinnamate groups promoted the adsorption of pullulan onto all surfaces and the adsorption onto hydrophobic surfaces was significantly greater than onto hydrophilic surfaces. SPR and QCM-D results showed that P3MC and P4CC also formed highly hydrated layers (70 to 90% water by mass) with low shear viscosities and elastic shear moduli. Finally, cellulose adsorption and activity on pullulan cinnamate (PC) and cellulose blend films were studied via QCM-D and in situ atomic force microscopy (AFM). The hydrophobicity of PC surfaces was controlled by adjusting the degree of cinnamate substitution per anhydroglucose unit (DSCinn). It was found that cellulase showed weak adsorption onto low DSCinn PC surfaces, whereas cellulase adsorbed strongly onto high DSCinn PC surfaces, a clear indication of the role surface hydrophobicity played on enzyme adsorption. Moreover, cellulase catalyzed hydrolysis of cellulose/PC and cellulose/polystyrene (PS) blend surfaces was studied. The QCM-D results showed that the cellulase hydrolysis rate on cellulose in cellulose/PC blend surfaces decreased with increasing DSCinn. AFM images revealed smooth surfaces for cellulose/PC (DSCinn = 0.3) blend surfaces and laterally phase separated morphologies for cellulose/PC (DSCinn ≥ 0.7) blend surfaces. The combination of QCM-D and AFM measurements indicated that cellulase catalyzed hydrolysis was strongly affected by surface morphology. The cellulase hydrolysis activity on cellulose in cellulose/PS blend surfaces was similar with cellulose/PC blend surfaces (DSCinn ≥ 0.7). These studies showed self-assembly of macromolecules could be a promising strategy to modify material surfaces and provided further fundamental understanding of adsorption phenomena and bioactivity of macromolecules at liquid/solid interfaces. / Ph. D.

Surface plasmon resonance has become a widely investigated phenomenon in the past few years. Initially descriptive of light interactions with metallic films, research has branched out to encompass the nanoparticles as well. Generation of the maximum surface plasmon resonance for nanostructures is based on the resonance condition that the oscillatory behavior of the 'free' electrons on the surface of the particle become equivalent to the frequency of the excitation light; for films this required a specific geometry. Metallic nanoparticles have also interested researchers because of their unique optical properties. Depending on the metal, observations of quenching as well as fluorescence enhancement have been reported. Based on the phenomenon of surface plasmon resonance as well as the properties of metallic nanoparticles, this research reports the interaction of gold and silver nanoparticles in an aqueous dye solution. Our research is the basis for developing an optical sensor used for water treatment centers as an alarm mechanism. Due to the inefficiency of the fluorophore used in similar optodes, sufficient fluorescence was not obtained. With the addition of the nanoparticles, we hoped to observe the transfer of energy from the nanoparticle to the fluorophore to increase the overall intensity, thereby creating a sufficient signal. Using the excitation theories discovered by Raman, Mie, and Forster and Dexter as our foundation, we mixed a strongly fluorescent dye with gold nanoparticles and aagain with silver nanoparticles. After taken measurements via fluorescence spectroscopy, absorption spectroscopy, and photoluminescence excitation, we observed that the silver nanoparticles seemed to enhance the fluorescence of the dye while the gold nanoparticles quenched the fluorescence. / Master of Science

Metallic nanoparticles (NPs) are exploited for their ability to interact with organic compounds and to increase significantly the fluorescence intensity and the photostability of many fluorescent dye molecules. Metal enhanced fluorescence (MEF) is therefore widely investigated for biosensing applications. When used in immunoassays, silver island films (SIFs) could augment the fluorescence intensity of fluorescein by a factor of seventeen; SIFs were also able to double or triple the emission intensity of cyanine dyes which are commonly used in (deoxyribonucleic acid) DNA microarrays. The emission intensity of indocyanine green — widely used as a contrast agent in medical imaging — was about twenty times higher in the proximity of SIFs. This enhancement phenomenon — due to the surface plasmon polaritons associated with the metallic NPs — can be explained by energy transfer from the metal NPs to the fluorescent dye molecules or by a modified local electromagnetic field experienced by the fluorophores in the vicinity of metal surfaces. Our research focused on the optical characterization of colloidal gold-silver alloy NPs containing different ratios of gold and silver (Au_1.00-Ag_0.00, Au_0.75-Ag_0.25, Au_0.50-Ag_0.50, and Au_0.25-Ag_0.75), as well as their interaction with three fluorophores: rose bengal, rhodamine B, and fluorescein sodium. Depending upon the dye quantum yield and its concentration in solution, enhancement or quenching of fluorescence was obtained. Thus, a three to five times increase in fluorescence intensity was observed in a 2.0 mM solution of rose bengal with all nanoalloys, a slight enhancement of fluorescence (1.2 – 1.6 times) was noticed in a 0.13 mM solution of rhodamine B with all four types of NPs, and fluorescence quenching occurred in all the fluorescein-NP solutions regardless of the dye concentration. / Master of Science

Surface plasmon resonance (SPR) is a label-free, versatile and highly sensitive method for studying molecular interactions in real time. It is widely used by industry and academia alike in fields ranging from Alzheimer’s disease research to detection of heavy metals. In this review, studies published during the last 10 years using Biacore or other SPR instruments were compiled and compared. Trends were also identified in the field. Amine coupling was found to be the most common ligand strategy for proteins, and most SPR research related to the field of medicine. Furthermore, three main purposes of an SPR experiment were identified: To determine the affinity between a pair of molecules, kinetics between a pair of molecules or to detect a certain molecule in a solution. The results presented are often related to these three purposes, and are most often presented and evaluated in terms of kinetic, affinity and sensitivity constants. SPR can be used for studying a broad range of molecular interactions, and an overview was obtained by dividing up the field into different parts based on molecular interactions and SPR methods. The study of molecular interactions using SPR was divided into protein-protein interactions (PPIs), antibody-antigen, protein-biomolecule interactions, interactions between proteins and small molecules, and non-conventional SPR methods. Non-conventional SPR methods include localized surface plasmon resonance (LSPR) and SPR imaging (SPRi), which are both based on the same optical sensing principles as SPR.

Noble metal nanostructures possess unique properties including large near-field enhancement and strong light scattering and absorption due to their plasmon resonance - the collective coherent oscillation of the metal free electrons in resonance with the electromagnetic field of light. The effect of nanostructure size, shape, composition, and environment on the plasmon resonance frequency and plasmonic enhancement is well known. In this thesis, we describe the effect of inter-particle coupling in assembled plasmonic nanostructures on their radiative and non-radiative properties. When metal nanoparticles assemble, plasmon oscillations of neighboring particles couple, resulting in a shift in the plasmon resonance frequency. Our investigation of plasmon coupling in gold nanorods shows that the coupling between the plasmons is "bonding" in nature when the plasmon oscillations are polarized along the inter-particle axis, whereas an "anti-bonding" interaction results when the polarization is perpendicular. We studied the distance-dependence of plasmon coupling using electrodynamic simulations and experimental plasmon resonances of lithographically fabricated gold nanoparticle pairs with systematically varying inter-particle separations. The strength of plasmon bonding, reflected by the fractional plasmon shift, decays near-exponentially with the inter-particle separation (in units of particle size) according to a universal trend independent of the nanoparticle size, shape, metal type, or medium. From the universal scaling model, we obtain a "plasmon ruler equation" which calculates (in good agreement with the experiments of Alivisatos and Liphardt) the inter-particle separation in a gold nanosphere pair from its plasmon resonance shift, making it applicable to the determination of inter-site distances in biological systems. Universal size-scaling is valid also in the metal nanoshell structure, a nanosphere trimer, and pairs of elongated nanoparticles, thus making it a generalized fundamental model, which is useful in optimizing plasmon coupling for achieving tunable plasmon resonances, enhanced plasmonic sensitivities, and large SERS cross-sections. Ultrafast laser pump-probe studies of non-radiative electronic relaxation in coupled metal nanospheres in aggregates and in gold nanospheres conjugated to thiol SAMs are also reported. We also show that the relative contribution of scattering (radiative) to absorption (non-radiative) part of the plasmon relaxation, respectively useful in optical and photothermal applications, can be increased by increasing the nanostructure size.

With a population both increasing in number and age, comes a need for new diagnostic tools in the healthcare system, capable of diagnosing and monitoring multiple disorders in a cheap and effective way to provide personalised healthcare. Multiplex label-free biosensors have the potential to rejuvenate the current system. This thesis details the assessment of an ‘in house’ built labelfree array screening technology that has potential to be a point-of-care diagnostic for personalised medicine – the Array Reader. The performance of the Array Reader platform is considered in detail and optimised for both antibody and protein screening arrays. A Global Fit protocol is developed to extract kinetic constants for all protein-protein interactions, assuming a Langmuir adsorption binding model. Standard operating procedures are developed to provide optimised dynamic range, sensitivity, reproducibility and limit of detection of immuno-kinetic assay. A new antibody bio-stack signal amplification strategy is formed, improving the detection limit 60-fold. As a consequence, the bio-stack resulted in a novel method for determining the plasmon field penetration depth, defining the assay sensing volume at the nanoparticle surface. Antibody screening arrays were investigated with an IgG quantification assay to determine total IgG content from serum samples. It relied on the ability of protein A/G to bind antibodies via the Fc region. Specific antigens were used to measure the binding properties of the antibody Fab region. By characterising both regions, we have gained insight into the overall ability of an antibody to trigger an immune response. Protein screening assay were investigated targeting C-reactive protein (CRP), a marker of inflammation. The assays performance characteristics compared favourably with clinically used CRP assays. Finally, an antibody screening array was developed to assess the efficacy of a vaccine against Yersinia pestis in a non-human primate model. The vaccine screening array is an excellent example of the versatility of the platform and just one of many possible applications for the future.

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.

The outbreak of pandemic influenza and its ability to spread rapidly makes it a severe threat to public health. Antiviral drugs such as oseltamivir (Roche’s Tamiflu™) and zanamivir (GlaxoSmithKline’s Relenza™) are neuraminidase (NA) inhibitors (NI), which bind more tightly to NA than its natural substrate, sialic acid. However, the virus can acquire resistance to antiviral drugs by developing single point mutations (such as H274Y) in the target protein. Thus in some cases the drugs may not be as effective as expected. The high level of inconsistency exhibited by fluorometric assays and the short half-life of the chemiluminescent assay for monitoring drug resistance lead to the need for a simple, label-free, reliable assay. To address this problem, this work focused on three main objectives: 1) to determine the binding affinities of two common anti-viral drugs (oseltamivir and zanamivir) against the influenza NA wild type and drug resistant mutants using bioinformatics software Schrodinger Suite™ 2010. 2) To develop a reliable label-free, real-time, surface plasmon resonance (SPR) assay to measure the binding affinity between influenza viral coat protein neuraminidase (wild type and mutant) and anti-viral drugs. 3) To develop an SPR inhibition assay to quantitatively compare the interactions of sialic acid, zanamivir and oseltamivir with the viral coat protein neuraminidase (wild type and mutant). The entire docking process was carried out using Schrödinger Suite™ 2010. The 2009 pandemic H1N1 neuraminidase (PDB: 3NSS) was used throughout the docking studies as the wild type structure. Five mutants (H274Y, N294S, H274N, A346N and I222V) and three ligands (sialic acid, oseltamivir and zanamivir) were built using the maestro module. The grid-based ligand docking with energetics (GLIDE) module and induced fit docking (IFD) module were used for docking studies. The binding affinities, Gibbs free energy change (∆G) and molecular mechanics-generalized born energy/ solvent accessible area (MM-GB/SA) values for wild-type NA interactions show that both the antiviral drugs studied interact strongly with the wild-type protein. The ∆G values for all antiviral interactions with mutant NA forms were reduced in magnitude, thereby indicating that they are less favourable than interactions with the wild-type protein. A similar trend was observed with MM-GB/SA results. Amongst all of the computed values, MM-GB/SA was the closest to the experimental data. In several cases, the interactions between the anti-viral drugs and NA mutants were markedly less favourable than those between sialic acid and the same mutants, indicating that these mutations could confer anti-viral resistance. Influenza NA wild-type and H274Y mutant were expressed in baculovirus expression system (BVES) in insect cells. The expressed proteins were partially purified using the standard purification techniques of anion exchange and size exclusion chromatography (SEC). A fluorometric activity assay was performed on the recombinant proteins. Both the wild type and the mutant showed similar level of activities. In addition, the recombinant NAs were used in an inhibition assay. Oseltamivir was found to be sensitive to wild type protein (IC50 = 0.59 nM) and resistant to the H274Y mutant protein (IC50 = 349.43 nM). On the other hand, zanamivir was sensitive to both wild type (IC50 = 0.26 nM) and the H274Y mutant (IC50 = 0.44 nM). This indicated that zanamivir was a more potent inhibitor than oseltamivir. These findings were in good agreement with the literature. An SPR assay for accurate monitoring of influenza antiviral drug resistance was developed. A spacer molecule (1, 6- hexanediamine) was site-specifically tethered to the inert 7-hydroxyl group of zanamivir. The tethered zanamivir was immobilized onto an SPR GLC chip to obtain a final immobilization response of 431 response units (RU). The reference subtracted binding responses obtained for NA wild-type and H274Y mutant were analysed using the ProteOn Manager™ Software tools. The SPR curves were fitted to a simple Langmuir 1:1 model with drift to obtain association rate constant (ka) and dissociation rate constants (kd). The relative binding values obtained from literature and the current SPR assay (1.9 and 1.7 respectively) suggested that the current SPR assay yielded similar results to the existing labelled enzymatic assay. In addition, an SPR inhibition assay was developed. The calculated IC50-spr values were compared and it was observed that oseltamivir was sensitive to wild type protein (IC50-spr = 7.7 nM) and resistant to the H274Y mutant protein (IC50-spr = 256 nM). On the other hand, zanamivir was sensitive to both wild type (IC50-spr = 2.16 nM) and the H274Y mutant (IC50-spr = 2.4 nM). Sialic acid was also found to be sensitive to both wild type (IC50-spr = 5.5 nM) and H274Y mutant (IC50-spr = 3.25 nM). In the cases studied, the viral proteins remained sensitive to sialic acid, consistent with retention of virulence of these mutant strains. It was concluded that zanamivir is a more potent inhibitor than oseltamivir for treating the H274Y mutant. Comparison of the SPR inhibition results with the docking results revealed a similar trend. The wild-type NA and H27Y mutant retained binding affinity for sialic acid and zanamivir. Oseltamivir showed a significant decrease in binding affinity for the H274Y mutant compared with the wild-type. This was because of the disruption of the salt bridge formation within NA that was vital for oseltamivir activity. To my knowledge, this is the first SPR biosensor assay developed to monitor influenza antiviral drug resistance. There is a tremendous scope to extend this study to more mutants and new antiviral drugs. This could pave the way for a reliable SPR biosensor assay to replace low consistency labelled enzymatic assays.

Proliferating cell nuclear antigen (PCNA) is an essential protein in the cell. It is involved in transcription and many types of DNA repair and replication. Homologues of this protein are found in all orders of life. The high level of conservation and essential nature of PCNA infers that it may be a potential drug target for anti-caner drugs in humans and also a potential anti-parasitic target. X-ray structures of PCNA from Homo sapiens (Hs), Schizosaccharomyces pombe (Sp) and Leishmania major (Lm) are now available and can be used as a template for structure based drug design. In this work PCNA from these three species have been prepared in milligram quantities for biochemical and biophysical studies. The previously unknown structure of LmPCNA has been solved in an uncomplexed form and also complexed with a dodecapeptide to a resolution of 3.0Å. A comparison of PCNA structures and their peptide complexes for the three species identifies structural differences which may be relevant in analysing thermodynamic contributions of binding. All eukaryotic PCNA molecules exist as ring shaped trimers which form around DNA. In this work the oligomeric state of LmPCNA has been determined to be hexameric both in solution and in the crystal. It has also been hypothesised that HsPCNA is hexameric however these would seem to form hexamers in which the trimeric rings associate “back-to-back” while LmPCNA trimers would seem to associate “face-to-face”. The binding affinities for these three PCNAs have been determined with a selection of peptides derived from the Hs p21 protein. This work has shown, using a selection of different techniques including Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC) and Dynamic Scanning Fluorimetry (DSF); that HsPCNA and SpPCNA have similar affinities for a 12mer peptide (Kd of ~1μM) however LmPCNA shows significantly weaker interactions (Kd of ~10μM). This is most likely due to divergence in the sequence and structure of LmPCNA. A systematic investigation by SPR on the effect of peptide linker length on binding has been carried out using a series of synthesised peptides with different lengths of chemical spacer. The series of streptavidin immobilised peptides show that longer spacers are required for the recovery of the PCNA peptide binding affinity. The results presented in this work indicate that a linker length of at least 20Å is required for measurable protein binding activity. This interaction is improved with longer peptide spacers.

GABAB receptors are G-protein coupled receptors for the major inhibitory neurotransmitter in the mammalian central nervous system, γ-aminobutyric acid (GABA). The receptor is linked to a variety of disorders including epilepsy, pain, spasticity, drug addiction and cognitive impairment and is, therefore of major importance for drug discovery. The most abundant receptor isoforms GABABR1a and R1b differ by the presence in R1a of a pair of Nterminal extracellular complement control protein modules (CCP1 and CCP2) which - in other proteins - are generally involved in mediating specific protein-protein recognition. The CCP1 module contains disulphides but is natively disordered. In the current work, the yeast two-hybrid system was used to confirm an interaction of CCP1 of GABABR1a with the extracellular protein fibulin-2. Further work with the yeast twohybrid system extablished the novel interaction of the abundant extracellular matrix protein laminin, with GABABR1a CCP1, via its laminin globular (LG) domains. The laminin interaction was further characterised by surface plasmon resonance, demonstrating that several different domains are involved in the binding to the GABAB receptor CCPs. The primary binding site is located on laminin α5 LG4-5, but the E10 domains of the β1 chain and LG1-3 on α1 may also be involved. The pharmacological properties of the GABABR1a and R1b isoforms were studied by transient expression in Xenopus laevis oocytes. It was demonstrated that the agonist baclofen, as well as the antagonist CGP55845, appear to be more potent at GABABR1b compared to GABABR1a. Intriguingly, when recorded in the precence of laminin, GABABR1b/R2 expressing oocytes exhibited an increased baclofen-evoked response while the response in GABABR1a/R2 was completely abolished. In conclusion, the work demonstrates that laminin is a binding partner for GABABR1a CCPs. Such an interaction between the metabotropic GABA receptor and the extracellular matrix may lie behind the recently reported roles of GABA in neuronal migration and the laying down of neuronal circuitry during the development of parts of the central nervous system.