In this dissertation, I have verified that the striking purple color of the intermetallic compound AuAl₂, also known as purple gold, originates from surface plasmons (SPs). This contrasts to a previous assumption that this color is due to an interband absorption transition. The existence of SPs was demonstrated by launching them in thin AuAl2 films in the Kretschmann configuration, which enables us to measure the SP dispersion relation. I observed that the SP energy in thin films of purple gold is around 2.1 eV, comparable to previous work on the dielectric function of this material. Furthermore, SP sensing using AuAl₂ also shows the ability to measure the change in the refractive index of standard sucrose solution. AuAl₂ in nanoparticle form is also discussed in terms of plasmonic applications, where Mie scattering theory predicts that the particle bears nearly uniform absorption over the entire visible spectrum with an order magnitude higher than a lightabsorbing carbonaceous particle. The second topic of this dissertation focuses on plasmon enhanced fluorescence in gold nanoparticles (Au NPs). Here, I investigated the distance-dependent fluorescence emission of rhodamine green 110 fluorophores from Au NPs with tunable spacers. These spacers consist of polyelectrolyte multilayers (PEMs) consisting of poly(allylamine hydrochloride) and poly(styrene sulfonate) assembled at pH 8.4. The distance between Au NPs and fluorophores was varied by changing the ambient pH from 3 to 10 and back, which causes the swelling and deswelling of PEM spacer. Maximum fluorescence intensity with 4.0-fold enhancement was observed with 7-layer coated Au NPs at ambient pH 10 referenced to pH 3. The last topic of this dissertation examines a novel approach to assemble nanoparticles, in particular, dimers of gold nanospheres (NSs). 16 nm and 60 nm diameter NSs were connected using photocleavable molecules as linkers. I showed that the orientation of the dimers can be controlled with the polarization of UV illumination that cleaves the linkers, making dipolar patches. This type of assembly provides a simple method with potential applications in multiple contexts, such as biomedicine and nanorobotics. / PHD / This dissertation covers three related topics. The first is an investigation of the optical properties of the unusually colored purple gold, which is a blend of gold and aluminum with the chemical formula is AuAl₂. This compound is interesting in that the origin of this color is different from most other metals. In the case of gold, for example, the metal gold is yellow color by absorbing the blue component from white light, leaving behind yellow color reflected light. The blue light is absorbed by electrons that change their state from a lower energy to a higher one. In purple gold, the color results from a different phenomenon known as “surface plasmons.” Surface plasmons are waves consisting of many electrons that move back and forth near an interface between a metal and an electrical insulator. The energy of surface plasmons in purple gold is low and corresponds to the purple color in this compound. Recently, published theoretical work supports the possibility of surface plasmons in purple gold. In this dissertation, I experimentally verify the presence of surface plasmons in purple gold. To launch surface plasmons, light was reflected off of a purple gold film deposited on the hypotenuse of a prism with varying angles of incidence. Surface plasmons can be observed by the sudden dimming of reflected light. From this, I was able to extract the surface plasmon dispersion relation, which is the relation between the inverse of the wavelength and the energy of the surface plasmons. In addition, I computed the light absorption properties of purple v gold when it is used in a nanoparticle form. The computational result showed that small purple gold nanoparticles absorb light very well, which may be useful in photothermal cancer therapy and solar steam generation. The second dissertation topic comprises a study of fluorescent molecules. These are compounds that reemit light with a different and redder color than the color of the light that illuminates them. In this experiment, green fluorescent molecules were placed near the surface of gold nanoparticles to observe how the brightness of the light emission is affected by the distance between the molecule and the metal. The underlying mechanism is based on localized surface plasmon resonances in gold nanoparticles. Localized surface plasmon resonances are waves consisting of many electrons that oscillate inside the particle, and they only occur when light at certain frequency illuminate the particle. On the resonance, the particle also exhibits the brighter light around the particle’s surface but the dimmer light away from the particle’s surface. The light enhancement from the particle can change the light emission of the fluorescent molecules. If the fluorescent molecules were placed in the range of localized surface plasmon resonances, the light emission is increased owing to the brighter light from the particle. However, if the fluorescent molecules were placed further away from the range of localized surface plasmon resonances, the light emission is decreased owing to the dimmer light from the particle. The distance between the surface of gold nanoparticle and the fluorescent molecules was varied by wrapping the gold particles with ultra-thin films of different plastic polymers before attaching fluorescent molecules to the surface of the films. These polymer films have the property that they swell and shrink when the acidity and basicity of the solution of gold particles changes, which allows me to vary the distance between the gold particles and fluorescent molecules. The results showed that the observed light gets dimmer when the solution is more acidic. On the other hand, the brighter light is noticed when the solution is more basic, and this observation is repeatable many times. Moreover, my work differs from other published works vi in that the particles with the polymer films are more robust and stable than the other particles. This allows more design flexibility and suggests applications in biomedical or environmental research where the particles can be used to locally measure properties, such as acidity in confined spacers such as living cells. It may be possible to use this technique for tumor cells in our body or toxic pollutants in the air or water. The last dissertation topic involves assembling nanoparticles to build them into larger structures. In this experiment, I fabricated particle dimers that consisted of two gold nanospheres of different sizes. They were attached together by using small molecules that are sensitive to ultraviolet (UV) light, where these molecules allow small gold nanospheres to be attached to large gold nanospheres only in those locations on the large nanospheres that have been illuminated with a sufficient amount of UV light. To achieve this alignment, UV light with a linear polarization (a specific electric field direction) was used to select the area on the large nanospheres where the UV light was particularly intense and therefore able to break the molecules, leaving positively charged surface patches on the spheres. This results in the electrostatic attraction between the positive patches on the large gold nanospheres and the negatively charged small gold nanospheres. With this method, I was able to make dimers of nanospheres in a preferred alignment by changing the polarization of UV light. The experimental results showed a good yield of dipolar patches, which allows multifunctional nanostructures with applications in nanomedicine, optical sensing, nanoelectronics, etc.

We have developed a general theory of the plasmonic enhancement of many-body phenomena resulting in a closed expression for the surface plasmon-dressed Coulomb interaction. It is shown that this interaction has a resonant nature. We have also demonstrated that renormalized interaction is a long-ranged interaction whose intensity is considerably increased compared to bare Coulomb interaction over the entire region near the plasmonic nanostructure. We illustrate this theory by re-deriving the mirror charge potential near a metal sphere as well as the quasistatic potential behind the so-called perfect lens at the surface plasmon (SP) frequency. The dressed interaction for an important example of a metal–dielectric nanoshell is also explicitly calculated and analyzed. The renormalization and plasmonic enhancement of the Coulomb interaction is a universal effect, which affects a wide range of many-body phenomena in the vicinity of metal nanostructures: chemical reactions, scattering between charge carriers, exciton formation, Auger recombination, carrier multiplication, etc. We have described the nanoplasmonic-enhanced Förster resonant energy transfer (FRET) between quantum dots near a metal nanoshell. It is shown that this process is very efficient near high-aspect-ratio nanoshells. We have also obtained a general expression for the force exerted by an electromagnetic field on an extended polarizable object. This expression is applicable to a wide range of situations important for nanotechnology. Most importantly, this result is of fundamental importance for processes involving interaction of nanoplasmonic fields with metal electrons. Using the obtained expression for the force, we have described a giant surface-plasmoninduced drag-effect rectification (SPIDER), which exists under conditions of the extreme nanoplasmonic confinement. Under realistic conditions in nanowires, this giant SPIDER generates rectified THz potential differences up to 10 V and extremely strong electric fields up to 10^5-10^6 V/cm. It can serve as a powerful nanoscale source of THz radiation. The giant SPIDER opens up a new field of ultraintense THz nanooptics with wide potential applications in nanotechnology and nanoscience, including microelectronics, nanoplasmonics, and biomedicine. Additionally, the SPIDER is an ultrafast effect whose bandwidth for nanometric wires is 20 THz, which allows for detection of femtosecond pulses on the nanoscale.

Wavelength-tunable optical response from solid-state optoelectronic devices is a desired feature for a variety of applications such as spectroscopy, laser emission tuning, and telecommunications. Nonlinear optical response, on the other hand, has an important role in modern photonic functionalities, including efficient frequency conversions, all-optical signal processing, and ultrafast switching. This study presents the development of optical devices with wavelength tunable or nonlinear optical functionality based on plasmonic effects. For the first part of this study, widely wavelength tunable optical bandpass filters based on the unique properties of long-range surface plasmon polaritons (LR SPP) are presented. Planar metal stripe waveguides surrounded by two different cladding layers that have dissimilar refractive index dispersions were used to develop a wide wavelength tuning. The concept was demonstrated using a set of index-matching fluids and over 200nm of wavelength tuning was achieved with only 0.004 of index variation. For practical application of the proposed concept, a thermo-optic polymer was used to develop a widely tunable thermo-optic bandpass filter and over 220 nm of wavelength tuning was achieved with only 8 ºC of temperature variation. Another novel approach to produce a widely wavelength tunable optical response for free-space optical applications involves integrating plasmonic metasurfaces with quantum-electronic engineered semiconductor layers for giant electro-optic effect, which is proposed and experimentally demonstrated in the second part of this study. Coupling of surface plasmon modes formed by plasmonic nanoresonators with Stark tunable intersubband transitions in multi-quantum well structures induced by applying bias voltages through the semiconductor layer was used to develop tunable spectral responses in the mid-infrared range. Experimentally, over 310 nm of spectral peak tuning around 7 μm of wavelength with 10 ns response time was achieved. As the final part of this study, highly nonlinear metasurfaces based on coupling of electromagnetically engineered plasmonic nanoresonators with quantum-engineered intersubband nonlinearities are proposed and experimentally demonstrated. In the proof-of-concept demonstration, an effective nonlinear susceptibility over 50 nm/V was measured and, after further optimization, over 480 nm/V was measured for second harmonic generation under normal incidence. The proposed concept shows that it is possible to engineer virtually any element of the nonlinear susceptibility tensor of the nonlinear metasurface. / text

Title: Surface plasmons in optical microstructures and their sensor applications Author: Pavel Adam Institute: Institute of Photonics and Electronics AS CR, v.v.i., Department of Optical Sensors Supervisor of the doctoral thesis: doc. Ing. Jiří Homola, CSc., DSc. Abstract: This work is focused on the study of surface plasmon resonance (SPR) sensor platforms based on wavelength division multiplexing (WDM) of multiple surface plasmons (SPs). These sensors are based on advanced diffraction gratings supporting either conventional or Bragg-scattered SPs, which are simultaneously excited at different wavelengths. These SPs are studied both analytically and numerically using rigorous coupled-wave analysis and an integral approach. WDM of two and three SPs is presented and followed by the method for the analysis of the resolution, noise and cross-sensitivity. This method is employed to analyze the ability of different SPR sensor platforms (supporting WDM of two SPs) to discriminate refractive index (RI) changes in a thin layer at the sensor surface from background RI changes. The WDM SPR sensors based on advanced diffraction gratings prepared by interferometric holography are developed and tested in a model biosensing experiment consisting of the layer-by-layer growth of protein multilayers. The linear WDM of two...

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.

The terahertz regime has until recently been some what neglected due to the difficulty of generating and measuring terahertz radiation. Terahertz time domain spectroscopy has allowed for affordable and broadband probing of this frequency regime with phase sensitive measurements (chapter 3). This thesis aims to use this tool to add to the knowledge of the interactions between electromagnetic radiation and matter specifically in regard to plasmonics. This thesis covers several distinct phenomena related to plasmonics at terahertz frequencies. The generation of terahertz radiation from metal nanoparticles is first described in chapter 4. It is shown that the field strength of the plasmon appears to relate to the strength of the generated field. It is also shown that the power dependence of the generated terahertz radiation is not consistent with the optical rectification description of this phenomenon. An alternative explanation is developed which appears more consistent with the observations. A simple model for the power dependence is derived and compared to the experimental results. In chapter 5 the parameters that make good plasmonic materials are discussed. These parameters are used to assess the suitability of semiconductors for terahertz surface plasmon experiments. The Drude permittivity of InSb is measured here, leading to a discussion of terahertz particle plasmons in chapter 6. Finite element method modelling is used to show some merits of these over optical particle plasmons. This also includes a discussion of fabrication methods for arrays of these particles. Finally, chapter 7 is a discussion of so called spoof surface plasmons. This includes some experimental work at microwave frequencies and an in depth analysis of open ended square hole arrays supported by model matching method modelling. Perfect endoscope effects are discussed and compared to superlensing. The thesis ends with a brief conclusions chapter where some of the ideas presented are brought together.