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The sensing applications based on surface plasmon resonance (SPR) have been developed rapidly in recent decades. Much attention has been paid for its real time sensing and label-free detection ability. A commercial success of the application on SPR technique is the BiacoreTM SPR systems developed by General Electric (GE) Heathcare. It has been widely used in hospitals and research institutes in various fields from biomedical/chemical reaction detection to the environment monitoring and food safety controlling. However, this prism-based SPR sensing system suffers from its bulk size design, moderate sensitivity and high cost. Further, the invention of optical fibre accelerated the development of SPR sensors, named as fibre optical plasmon sensor (FOPS) by utilizing the conventional optical fibre. It has been proven to be a potentially promising tool for the realisation of small-size sensor with excellent sensing performance and the capability of remote sensing. The FOPS has a significant importance on the development of SPR sensors in various ways. Later on, the emergence of photonic crystal fibre (PCF)with FOPS has opened up a new era of sensing technological innovations. The PCF based FOPS is now becoming a promising optical detection technique for studying label-free bio-molecular interactions in real time. In this thesis, the use of novel PCFs as a new class of optical fibre together with the SPR phenomenon for sensing application is presented and investigated in detail. By manipulating the structural parameters, the PCFSPR sensor can be optimized to achieve higher sensitivity, larger sensing range for better sensing performance and easy-fabrication. Additionally, efforts have been made to investigate the influence of the sub-peak for higher order SPR on the sensing performance. Moreover, a multi-sensing scheme by utilizing metallic nanowires in PCF may provide a new method for fast sensing. The key original contributions of this thesis include: 1. Modified D-shaped PQF-SPR Bio-sensor with Trapezoid Shaped Analyte Channel for High Refractive Index Liquid Analytes with Adjustable Liquid Height. To overcome those two disadvantages - low sensitivity and fabrication difficulty and to extend the sensing range, a six-fold trapezoidal channel (TC) PQF SPR bio-sensor for liquid bio-analyte sensing is demonstrated to investigate how the liquid height in the TC influences the sensing performance. The structure optimization of such sensor is presented for better sensitivity, sensing range and stability. The effective refractive indices and loss spectra peak differences acquired from the simulations of the designed TC-PQF sensor for a range of wavelengths have been solved and calculated thoroughly to evaluate its sensing performance. According to the numerical results, it shows a good sensitivity for the analyte RI ranges of 1.40 to 1.58 which is broad enough to cover most of the bio-samples. Besides, the result also proves that it is possible to control the sensor's detection range by adjusting the liquid analyte's height/thickness. The liquid analyte height/thickness is also found to influence the sensor's sensing performance and an inversely proportional relationship between the detection sensitivity and detectable analyte RI range has been concluded. This work helps us to gain an understanding on the effect of the quantity/height of the analyte of interest and the structural parameters on the sensing performance of such kind of PQF-SPR bio-sensor. Part of the work in the previous project is to form a side-channel into the fibre structure so that the liquid height can be varied in such channel. Although a detailed fabrication method of the proposed sensor with trapezoidal side-channel has been clearly discussed in chapter 3, a mature fabrication/manufacture process for this unique structure is not available on the market for massive production. Hence, from a commercial perspective, it would be economical and realizable to apply a common type of PCF, which is already available on the market as the substrate for sensors fabrication. Therefore, the six-fold hexagonal D-shaped PCF is introduced in the sensor design for easy-fabrication purpose. 2. The Influence of Secondary Plasmon Resonance Peak on the Sensing Performance of a Standard Six-fold D-shaped PCF-SPR Sensor. The side-polish technique currently is used to produce D-shaped PCF which has been widely studied by many researchers as a relatively mature post-processing method in lab. For this reason, it is necessary to introduce and investigate the sensing performance of such a six-fold D-shaped hexagonal PCF-SPR sensor. Its sensing details have been discussed and concluded in chapter 4 about the influence of different plasmonic resonance peaks between the fundamental mode and surface plasmons for a common six-fold hexagonal D-shaped PCF-SPR sensor. The causes of its sensing limitation is also investigated for better understanding of the modes in PCF. It is interesting that there is a secondary plasmon resonance peak existed that will have a significant impact on its sensing performance of system stability and accuracy, especially for analytes with high refractive index (RI = 1.42). 3. SPR Bio-sensor based on Dual Core D-Shaped PCF Embedded with Silver Nanowires for Multi-sensing. On the other hand, in order to increase the analyte sensing efficiency of the PCF-SPR biosensor, multi-sensing technique is brought into the sensor design as an efficient way to accelerate the detection processing time. In chapter 5, a novel dual-core D-shaped PCF based SPR biosensor with embedded silver nanowires is proposed for multi-sensing. This biosensor design can achieve two analytes detections simultaneously with high sensitivity and it also provides a broad sensing RI range which covers most of the refractive index values of already known viruses, proteins, DNAs (deoxyribonucleic acids)/RNAs (ribonucleic acids), antigens/antibodies. Moreover, with the innovative additional critical sensing channel, it helps the users to distinguish the analyte of interest with the corresponding sensing channel more easily. Whereas the other multi-channel sensing design can only show the sensing peaks in the loss spectra without any information about the location (channel) where the individual specific binding reaction takes place. 4. Modelling Instruction of a Common PCF-SPR Bio-sensor using COMSOL Multiphysics. As those three research projects are all based on the numerical analysis on COMSOL Multiphysics, a comprehensive modelling design flow based on COMSOL Multiphysics system about how to simulate a standard PCFSPR inner air hole metal-coating sensor was summarized and presented as an example to help anyone who will carry out the design process in a fast, simple and easy way (see Appendix A).

Significant improvement in using plasmonic nanostructures for practical colour filtering and multispectral imaging applications is achieved by exploiting the coupling of surface plasmon polaritons with dielectric optical cavity resonances within a hexagonal array of subwavelength holes in a thin CMOS-compatible metal-insulator-metal (MIM) stack. This architecture overcomes the limitations of all previously reported plasmonic colour filters, namely poor transmission and broad band-pass characteristic, effectively providing a compact approach to selectively filter light. The physical insights established from the electromagnetic near-field patterns and parametric simulation studies are used to accurately tailor the optical properties of the filters. Measured transmission efficiencies up to 60% and full-width at half-maximum between 45 nm and 55 nm along the entire visible spectrum are achieved - an impressive and unique combination of features that has never been reported before. The non-ideality of the fabrication at the nanoscale is addressed, the issues encountered highlighted and alternative solutions proposed and verified, demonstrating that the working principle of the MIM structure can be successfully extended to other materials and structural parameters.

Surface plasmon resonance (SPR) biosensors have attracted great attention in scientific research in the past three decades. Extensive studies on the immobilisation of biorecognition elements have been conducted in pursuit of higher sensitivity, but trialled formats have focussed on a thin layer modification next to the plasmon film, which usually requires in situ derivatization. This thesis investigates an 'off-chip' immobilisation strategy for SPR biosensing using silica particles and considers the implications of a particle-modified evanescent field on the signal amplitude and kinetics, for an exemplar affinity binding between immobilised IgG and its anti-IgG complement. Submicron silica particles were synthesized as carriers for the bio-recognition elements. They were then immobilised to form a sub-monolayer on the gold film of an SPR biosensor using two methods: thiolsilane coupling and physical adsorption aided by mechanical pressure. The bio-sensitivity towards an antigen/antibody interaction was lower than an SPR biosensor with an alkanethiolate SAM due to the difference in ligand capacity and position in the evanescent field. The binding kinetics of antigen/antibody pair was found to follow the Langmuir model closely in a continuous flow configuration but was heavily limited by the mass transport from the bulk to the sensor surface in a stop-flow configuration. A packed channel configuration was designed with larger gel particles as ligand carriers, packed on top of a gold film to create a column-modified SPR biosensor. This sensor has comparable bio-sensitivity to the previous sub-monolayer particle-modified systems, but the binding and dissociation of the analyte was heavily dependent on mass transport and binding equilibria across the column. A bi-directional diffusion mechanism was proposed based on a two-compartment mass transport model and the expanded model fitted well with the experimental data. The column-modified sensor was also studied by SPR imaging and analyte band formation was observed and analysed. Using the lateral resolution, a multiplexing particle column configuration was explored, and its potential in distinguishing a multicomponent analyte.

Tuberculosis affects millions of people worldwide every year. The current treatment for TB is divided into a regimen of both first- and second-line drugs, where first-line treatments are more tolerated and require shorter treatment lengths. With rising levels of resistance, alternative treatment regimes are urgently needed to fight this disease. Ethionamide, a second-line drug is administered as a prodrug which is activated in vivo by the enzyme EthA, which is in turn regulated by EthR. The disruption of the action of EthR could lead to novel therapeutics which could enhance the efficacy of ethionamide, and raise it to a first-line treatment. The work reported in this thesis examines the elaboration of three chemical scaffolds using fragment-based approaches to develop novel inhibitors capable of disrupting the EthR-DNA interaction. The first scaffold, 5-(furan-2-yl)isoxazole was investigated by fragment-merging approaches and produced compounds with the best of these having a KD of 7.4 uM. The second scaffold, an aryl sulfone was elaborated using fragment-merging strategies. This led to several modifications of the fragment, leading to several variants with KDs around 20 uM. With both of these series the affinity could not be improved below 10 uM and due to the synthetic complexity a further scaffold was prioritised. The third scaffold was explored was a 4-(4-(trifluoromethyl)phenyl)piperazine using fragmentgrowing from the NH of the piperazine to probe deeper into the EthR binding pocket. In addition to this, SAR around the 4-(trifluoromethyl)phenyl group was assessed to explore the interactions with EthR. These modifications led to compounds with nanomolar IC50s. A range of compounds were then screened by REMAssay to determine the boosting effect on ethionamide, and this identified compounds with up to 30 times boosting in the ethionamide MIC. The final chapter examines a concept where compounds were designed to exploit the dimeric nature of EthR by linking two chemical warheads with a flexible linker. These compounds are examined using mass spectrometry to investigate the stoichiometry of the interaction to provide insight into the binding of these extended compounds and exploring an alternative strategy to inhibit EthR. The work in this thesis demonstrated the successful use of fragment-based approaches for development of novel EthR inhibitors which showed significant ethionamide boosting effects.

Surface plasmons are collective oscillations of free electrons excited at a metal-dielectric interface by incident light. They possess a broad set of interesting properties including a high degree of tunability, the generation of strong field enhancements close to the metal's surface and high sensitivity to their adjacent dielectric environment. It is possible to enhance the sensitivity of plasmonic systems by using narrow plasmon resonances. In this thesis two approaches to narrowing surface plasmon resonances have been studied: diffraction coupling of localised surface plasmon resonances in gold nanoarrays and the use of graphene-protected copper thin films. Applications of these approaches in hybrid systems have been considered for modulation, waveguiding, biosensing and field enhancements. Arrays of gold nanostripes fabricated on a gold sublayer have been used to create extremely narrow plasmon resonances using diffraction coupling of localised plasmon resonances with quality factors up to a value of $Q \sim 300$, among the highest reported in the literature. The nanostructures were designed to give the narrowest resonance at the telecommunication wavelength of 1.5 µm, allowing for this array geometry to be used in hybrid systems for proof-of-concept optoelectronic devices. The gold nanostripe array was used in a hybrid nanomechanical electro-optical modulator along with hexagonal boron nitride (hBN) and graphene. The modulator was fabricated with an air gap between the nanoarray and the hexagonal boron nitride/graphene. Applying a gate voltage across the device moves the hBN towards the nanoarray, resulting in broadband modulation effects from the ultraviolet through to the mid-infrared dependant on the motion of the hBN instead of graphene gating. The deposition of a 400 nm hafnium(IV) oxide film on top of the gold nanoarray created a structure capable of guiding modes at 1.5 µm. The hybrid air-dielectric-stripe waveguide is capable of guiding modes over a distance of 250 µm. Copper thin films have stronger plasmon resonances and higher phase sensitivity than gold thin films. Transferring a graphene sheet on the copper prevents oxidation of the copper. A feasibility study of this hybrid system has shown that phase-sensitive graphene-protected copper biosensing can detect HT-2 mycotoxin with over four orders of magnitude greater sensitivity than commercially-available gold-based surface plasmon resonance biosensing systems. In summary, two methods of attaining narrow plasmon resonances have been demonstrated and their promise in modulation, waveguiding and biosensing have been demonstrated.

Heavy metal toxicity is a major environmental problem world-wide. Increased spreading and high concentration levels of the toxic heavy metals in water environments have posed a severe threat to human health and the ecosystem. Over the years, to improve the drinking water quality standards, safe threshold concentrations of these highly toxic pollutants are constantly being lowered by the governmental and environmental bodies. Current instrumental techniques used to detect these low levels of heavy metal ions are laboratory based, use sophisticated instruments, expensive, time consuming and require trained personnel. There is a constant need for the development of in-situ, rapid, highly sensitive and selective sensors to monitor the very low concentration levels. Various approaches for improving sensitivity and selectivity have been investigated over the years involving multiple detection techniques. In general, optical approaches provide higher sensitivity along with simplicity while electrochemical sensors provide better selectivity. In the last decade, nanomaterials have emerged as a key element in their sensitivity improvement. Combining all these advantages, a novel hybrid sensor has been envisaged integrating optical and electrical fields in addition to nanomaterials. This thesis reports investigations on enhancing the sensitivity/selectivity through optical, nanomaterials and electrochemical routes, and then integrating these to realise a hybrid sensor. A novel optical sensor has been developed using the phenomena of evanescent waves in optical fibre with dithizone to detect heavy metal ions. A U-bent sensor geometry has been investigated to enhance the optical sensitivity of the sensor through higher evanescent field near the surface. Further, optical field confinement to the surface has been investigated through thin film coating to improve the sensitivity. A new inverted trench design based sensor has been developed, and sensitivity enhancement has been achieved through this novel design and confirmed using modelling work accompanied by experimental results. Large surface to volume ratio of nanomaterials, such as ZnO nanowires, on the sensor surface can provide enhanced surface interactions leading to higher sensitivity. But, surfaces modified with ZnO nanostructures tend to be hydrophobic in nature. A new remote and non-contact method to tune the wettability of the ZnO nanostructures using LEDs has been developed. Higher sensitivity has been achieved by tuning the wettability of ZnO nanowires using the developed method. An electrochemical sensor has been developed in order to understand the potential effects of the electric field on the near surface molecular dynamics and thereby, effects on the optical detection. Effects of parameters such as deposition time, scan frequency, concentration, electrode materials and their surface area have been investigated to improve the sensitivity and selectivity. Multi-ions selectivity has been achieved by simultaneous detection of copper, mercury and lead ions in buffer solution. Higher sensitivity has been obtained by modifying the gold electrode using graphene flakes. Further, to integrate the optical field with this sensor to realize the hybrid sensor, thickness of the gold electrode has been optimised to allow the penetration of evanescent field onto the electrode surface. Under optimised conditions evanescent field resonantly couples to the surface plasmons of the gold electrode. Computational investigations have been carried out to study the effect of number of graphene layers on the sensitivity of the surface plasmon resonance (SPR) based optical sensor integrated with the electrochemical sensor. Preliminary investigations on the developed hybrid sensor show that the electric field complements the optical field. Investigations have shown that application of electric field enhances the sensitivity for optical detection by attracting more ions on the electrode and also, provides the multi-ion selectivity. These investigations have opened up new possibilities for the real-time monitoring of highly sensitive and selective molecular interactions, showing strong potential in a range of applications areas such as environmental sensing, biosensing and agricultural sensing.

The goal of this project is to study novel nanoscale excitation volumes, sensitive enoughto study individual chromophores and go on to study new and exciting self assemblyapproaches to this problem. Small excitation volumes may be engineered using light con-finement inside apertures in metal films. These apertures enhance fluorescence emissionrates, quantum yields, decrease fluorescence quenching, enable higher signal-to-noiseratios and allow higher concentration single chromophore fluorescence, to be studied byrestricting this excitation volume. Excitation volumes are reported on using the chro-mophore's fluorescence by utilising fluorescence correlation spectroscopy, which monitorsfluctuations in fluorescence intensity. From the correlation in time, we can find the res-idence time, the number of chromophores, the volume in which they are diffusing andtherefore the fluorescence emission efficiency. Fluorescence properties are a probe ofthe local environment, a particularly powerful tool due to the high brightness (quantumyield) fluorescent dyes and sensitive photo-detection equipment both of which are readilyavailable, (such as avalanche photodiodes and photomultiplier tubes). Novel materialscombining the properties of conducting and non-conducting materials at scales muchsmaller than the incident wavelength are known as meta-materials. These allow combi-nations of properties not usually possible in natural materials at optical frequencies. Theproperties reported so far include; negative refraction, negative phase velocity, fluorescenceemission enhancement, lensing and therefore light confinement has also been proposed tobe possible. Instead of expensive and slow lithography methods many of these materialsmay be fabricated with self assembly techniques, which are truly nanoscopic and otherwiseinaccessible with even the most sophisticated equipment. It was found that nanoscaled volumes from ZMW and HMMs based on NW arrays wereall inefficient at enhancing fluorescence. The primary cause was the reduced fluorescencelifetime reducing the fluorescence efficiency, which runs contrary to some commentatorsin the literature. NW based lensing was found to possible in the blue region of the opticalspectrum in a HMM, without the background fluorescence normally associated with a PAAtemplate. This was achieved using a pseudo-ordered array of relatively large nanowireswith a period just smaller than lambda / 2 which minimised losses. Nanowires in the traditionalregime lambda / 10 produced significant scattering and lead to diffraction, such that they werewholly unsuitable for an optical lensing application.

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.

Many studies have demonstrated a connection between the fibrillin matrix and TGFβ signalling, but at present the mechanistic basis for this link is unclear. An interaction between the C-terminus of Latent TGFβ Binding Protein 1 (LTBP1) and the N-terminus of fibrillin1 has previously been identified, and may have the potential to directly link the fibrillin matrix to TGFβ signalling. To investigate the structural basis for this interaction, several multi-domain fragments of fibrillin1 and LTBP1 were expressed prokaryotically and refolded in vitro. After initial characterisation to confirm folding, the structure, dynamics, and interdomain interactions of these fragments were investigated in more detail using NMR techniques. Domains in both LTBP1 and fibrillin1 appear to demonstrate folds consistent with homologous structures, and while the LTBP1 C-terminal cbEGF14-TB3-EGF3-cbEGF15 region contains many flexible linkers and few interdomain interactions, the fibrillin1 EGF2-EGF3-hyb1-cbEGF1 region appears rigid, with interfaces forming between all domains present. SPR studies were used to demonstrate binding between distinct LTBP1 and fibrillin fragments, suggesting interactions between multiple domains are involved in the LTBP1-fibrillin1 interaction. The binding sites involved were then mapped to specific residues using HSQC titration studies, and structural models for the LTBP1-fibrillin1 interaction were generated based on these data. Predictions from these models were used to target residues for site-directed mutagenesis, based on their potential involvement in salt bridges, and when certain residues were replaced with those of opposite charge, reductions in binding could be seen in the SPR assay. These key residues were consistent with a particular model of the LTBP1-fibrillin1 interaction, as derived from the HSQC titration data. The conservation of potential binding site residues through deuterostome evolution also supports an important biological role for the LTBP-fibrillin interaction.

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.

Optical trapping of metal nanoparticles investigates phenomena at the interface of plasmonics and optical micromanipulation. This thesis combines ideas of optical properties of metals originating from solid state physics with force mechanism resulting from optical trapping. We explore the influence of the particle plasmon resonance of gold and silver nanospheres on their trapping properties. We aspire to predict the force mechanisms of resonant metal particles with sizes in the Mie regime, beyond the Rayleigh limit. Optical trapping of metal nanoparticles is still considered difficult, yet it provides an excellent tool to investigate their plasmonic properties away from any interface and offers opportunities to investigate interaction processes between light and nanoparticles. Due to their intrinsic plasmon resonance, metal nanoparticles show intriguing optical responses upon interaction with laser light. These differ greatly from the well-known bulk properties of the same material. A given metal nanoparticle may either be attracted or repelled by laser light, only depending on the wavelength of the latter. The optical forces acting on the particle depend directly on its polarisability and scattering cross section. These parameters vary drastically around the plasmon resonance and thus not only change the magnitude but also the direction and entire nature of the acting forces. We distinguish between red-detuned and blue-detuned trapping, that is using a trapping wavelength shorter or longer than the plasmon resonance of the particle. So far optical trapping of metal nanoparticles has focussed on a wavelength regime far from the particle’s resonance in the infrared. We experiment with laser wavelengths close to the plasmon resonance and expand the knowledge of metal nanoparticle trapping available to date. Existing theoretical models are put to the test when we compare these with our real experimental situations.

Neurotensin (NT) is a tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. Neurotensin receptor 1 (NTS1), a high affinity receptor for NT, is a member of the GPCR superfamily and is a putative target for the treatment of conditions such as Schizophrenia, Parkinson’s Disease and drug addiction. Overexpression and purification are typically limiting steps in the high resolution structure determination of GPCRs. In this study, through the optimisation of the E.coli strain used for overexpression of rat NTS1 (NTS1) and the inclusion of phospholipids in the purification buffers to prevent delipidation, an approximate 3-fold improvement in active receptor yield was obtained relative to existing protocols. Preliminary electron microscopy (negative stain and cryo) confirmed a monodisperse receptor population. Purified NTS1 is now being produced at a sufficient level for high resolution structural studies, including 3D crystallisation and further electron microscopy studies. The existing construct for the expression of NTS1 in E.coli, termed NTS1B, was modified to contain a fusion to the genes encoding either the eCFP or eYFP fluorescent proteins. These constructs were used for the E.coli expression of NTS1 tagged with either fluorescent protein at the C-terminus. Tagged receptor was successfully expressed at levels of up to 0.29 ± 0.03 mg per l of culture. Successful purification and proteolytic removal of the MBP and TrxA-His10 fusion partners was achieved whilst retaining both fluorescence and ligand binding capability (Kd = 0.91 ± 0.17 nM). Purified, fluorescent receptor was reconstituted into brain polar lipid (BPL) liposomes in an active conformation which was both fluorescent and able to bind NT. Experimentation with alternative lipid compositions suggested that specific lipids are required in order to maintain ligand-binding activity. FRET between the eCFP- and eYFP-tagged receptors was observed in reconstituted samples. The FRET efficiency was comparable to that observed in vivo for other GPCRs, including the yeast α-factor receptor, which is believed to be dimeric. This suggests that NTS1 could also be multimeric. In contrast, no FRET was observed in detergent samples. Therefore, a functioning in vitro system has been developed which enables the study of NTS1 multimerisation in lipid bilayers and future studies will attempt to implement single molecule fluorescence techniques. In addition, fluorescent derivatives of NT were successfully synthesised and purified. Radioligand competition assays and fluorescence correlation spectroscopy (FCS) confirmed that the fluorescent peptides bound to purified NTS1 in specific competition with unlabelled NT. Surface plasmon resonance (SPR) was used to confirm the ligand binding activity of purified NTS1. A novel approach was utilised which involved the measurement of the binding of detergent-solubilised NTS1 to immobilised, N-terminally biotinylated NT on the sensor surface. The use of a rigorous control, which consisted of immobilised ‘scrambled sequence’ NT, demonstrated a specific interaction. Analysis of the kinetics revealed a multiphasic interaction with a Kd in the nanomolar range. In summary, improvements to the expression and purification of NTS1, the generation of fluorescent constructs as useful tools in the study of receptor multimerisation and the optimisation of lipid-reconstitution protocols have opened up several preliminary lines of study which show considerable potential for future research.

High-performing sensors have played a pivotal role in expanding our understanding of biological systems, disease diagnosis, environmental monitoring, and national security. The technical capability they provide has enabled us to obtain in-depth information and insights towards improving human health. One area of sensing that exemplifies this progress is the development of label free sensors which allow direct analysis of molecular interactions. Among these methods surface plasmon resonance (SPR) has emerged as a powerful, real-time detection technique for studies of biological interactions, drug discovery, and other important aspects that lead to new disease diagnosis. Through the implementation of new materials and methods SPR and other label-free sensors have expanded the range of analytes tested. This Dissertation aims to establish improvements in materials and methodologies through technology advancement for solving current sensor limitations. The work focuses on enhancing sensing signal while limiting the impact of nonspecific interactions on label-free methods, providing expanded molecular identity information, and overcoming challenges encountered when detecting small molecules. Chapters 2, 3, and 4 demonstrate advancements in unique biomimetic surfaces to enable the exploration of new biological systems as well as block nonspecific interactions. Chapter 2 focuses on a tethered membrane system to promote incorporation of relevant constituents into lipid bilayers without compromising membrane mobility property and drug delivery interactions. Chapter 3 employs a charged membrane to suppress nonspecific interactions and explores the working mechanism. Chapter 4 expands the capabilities of label-free sensing systems through development of curved membrane platforms that mitigate the decay limits through modeling of lipid distribution in vesicles. Chapter 5 exploits the plasmonic properties of SPR chips to enhance signals in matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) , which is further facilitated with development of machine learning models to identify bacterial species. In Chapter 6, the limitation of small molecule analysis with SPR is tackled by taking advantage of pressure effects to provide specific gas sensing. Each of these Chapters provides novel advancements in sensing capabilities by addressing performance-impairing limitations in label-free sensors. Research goals are achieved both from improvements to SPR systems and incorporation of other methodologies to augment SPR results.

Protein disulfide isomerase (PDI) is an endoplasmic reticulum (ER)-resident chaperone with oxidoreductase and isomerase activity. Unique to its normal function, PDI also appears to disassemble the A1 subunits of cholera toxin (CT) and heat-labile enterotoxin (LT). It does so using an unfolding mechanism that knocks the catalytic A1 subunit away from the rest of the holotoxin. Release of the A1 subunit is linked to the diarrheal diseases caused by V. cholerae and enterotoxicogenic E. coli (ETEC). Due to the previously established difference in disease potency between CT and LT, we investigated and established a distinction between the two toxins in their efficacy of disassembly by PDI. We further identified four amino acid differences between the CTA2 and LTA2 linkers, which connect the A1 and cell-binding B subunits of both toxins, as the basis for this difference. We believe these four amino acids result in changes to holotoxin architecture that lead to antiparallel binding of PDI to LT as opposed to CT, which translates to a loss of momentum for the physical disassembly of LT. We have shown this through algorithmic simulations of the binding event between PDI and either CT or LT. We hypothesized the unfolding mechanism of PDI, which dislodges the A1 subunit of both CT and LT, can also break down neurotoxic aggregates of β-Amyloid (AB) and α-Synuclein (AS). PDI is known to inhibit the aggregation of the amyloid proteins. We demonstrated here that PDI could also reverse oligomeric and post-oligomeric aggregates of AB and AS, respectively. Our work sheds light on the specifics of PDI's novel physical mechanism as well as introduce it as a possible therapeutic for both Alzheimer's and Parkinson's disease due to its unique ability to disaggregate early fibrillar structures of AS and AB proteins.

Recent advancements in life science research have greatly improved our understanding of various intricate biological systems, some of which are affiliated with complex diseases. While a great deal of progress has been made towards the comprehension of relevant biophysical interactions, many remain poorly understood. This is especially true for protein interactions involving the cellular membrane. Of the analytical strategies developed for investigating molecular interactions, surface plasmon resonance (SPR) provides marked technical advantages and has become a cornerstone for these studies. However, the SPR method is still facing challenges, from both technical aspects and investigation of diseases, which are the focus of this Dissertation. SPR is susceptible to misidentification in biological matrices, due to cross-reactive and nonspecific interactions. The lack of reliable curved biomimetic membrane platforms has limited the investigation on various disease related protein interactions. The presented Dissertation aims to provide solutions to these challenges through developing novel biomimetic membrane platforms, robust post data acquisitions analysis strategies, and antifouling protocols. Chapter 2 showcases the development of a self-assembled pseudo-myelin sheath microarray. The platform and developed antifouling protocol were shown to be capable of detecting three multiple sclerosis (MS) specific anti-ganglioside antibodies in 10 % serum. Chapter 3 expands and improves upon the platform presented in Chapter 2. The MS specific antibodies were detected at disease relevant concentrations, 3 ng/mL to 25 ng/mL, in undiluted human serum. Machine learning algorithms were applied to facilitate the differentiation and identification of the highly cross reactive analyte-antigen interactions in a clinical setting. In Chapter 4, the characterization and development of a tunable curved membrane mimicking platform is presented. Through the use of statistical analysis, simulations, and SPR, we have demonstrated that the curved membrane platform could be uniquely applied to quantify protein-membrane interactions that require curvature. Chapter 5 reports the use of the curved membrane platform for the investigation of bridging-integrator-1’s biophysical interactions, and their detection in biological fluids such as urine, which has been affiliated with muscular dystrophy. This study is the first quantitative evaluation of these interactions, and could aid in the diagnosis of muscular dystrophy. The works presented in this Dissertation have laid a solid foundation for advanced SPR biosensing that focuses on biomarker detection and disease diagnosis.

Colloidal copper phosphide (Cu3−xP) nanocrystals are attractive electronic materials due to their ability to support excess delocalized holes, with both synthesis and dynamic redox modulation allowing tuning of localized surface plasmon resonance (LSPR) absorption in the near-IR. This dissertation presents developments in the colloidal syntheses and post-synthetic redox chemistries of high-quality Cu3−xP nanoplatelets with the attainment of tunable nanocrystallite lateral sizes, LSPR energies, compositions, and optoelectronic properties. A one-pot, colloidal synthesis of Cu3−xP nanoplatelets with copper halide salt and aminophosphine in the presence of oleylamine is presented in Chapter 2. The reactivity of an in situ copper-phosphorus precursor is modulated by varying precursor composition and coordinating solvent ratio to tune nanocrystal lateral size. Oleylamine-to-aminophosphine molar ratio is found to alter particle nucleation activity and access atom-efficient size tuning. By modulating precursor reactivity, the syntheses presented in Chapter 2 are used to access nanocrystals with lateral sizes of 6.1–23 nm and LSPR energies of 709–861 meV. The low polydispersity, size- and LSPR-tunability and colloidal stability make aminophosphine-synthesized Cu3−xP nanocrystals promising candidates for investigations into factors governing their LSPR energy, around which the findings of the subsequent chapter are based. Three copper-coupled redox chemistries are presented in Chapter 3 which allow post-synthetic modulation of the delocalized hole concentrations and corresponding LSPR absorption in colloidal Cu3−xP nanocrystals. Changes in the structural, optical, and compositional properties are evaluated by powder X-ray diffraction, electronic absorption spectroscopy, 31P magic-angle spinning solid-state nuclear magnetic resonance spectroscopy and elemental analysis. The redox chemistries are shown to access nanocrystals with LSPR energies of 660–890 meV, a larger range than has been possible through synthetic tuning. The largest structural and LSPR modulation is achieved through the use of a divalent metal halide and trioctylphosphine, with the smallest reported unit-cell volume (295.2 Å3) and most downfield 31P chemical shift (+346 ppm) reported for P63cm Cu3−xP. In Chapter 4, the platform for systematic mechanistic investigations developed in Chapter 2 is extended to use copper phosphide as a model system for the study of aminopnictine precursor chemistry and the elucidation of metal pnictide nanocrystal formation mechanisms. Preliminary findings are presented for the investigation of the synthetic roles of copper salt counteranion and aminophosphine amide identity.

Arc is a repurposed retrotransposon Gag protein that transports mRNA between neurons via assembling into virus-like capsids. An Arc homolog, the HIV Gag, can selectively package the 5’ untranslated region (UTR) of its own genome RNA, leading to more efficient capsid assembly than other abundant host cytoplasmic RNAs. There is currently limited understanding on the mRNA selection mechanism during Arc capsid assembly. Investigating this mechanism can provide insight into Arc’s role in neurological function and impact within neurodegenerative diseases as well as for utilizing Arc as a potential delivery vehicle for therapeutic applications. Here, we investigate any preference that Arc protein potentially has towards binding the Arc 5’ UTR mRNA motif over other mRNA sequences. Dynamic light scattering (DLS) measurements and atomic force microscopy (AFM) confirmed in vitro Arc capsid assembly encapsulates any mRNA that are prominently present within the environment. A custom-made surface plasmon resonance (SPR) biosensor allowed for an in-depth study of biomolecular interactions between mRNA and Arc. The SPR biosensor results showcased Arc’s ability to nonspecifically bind to any mRNA sequence. Furthermore, it confirmed the existence of a selective mRNA binding site on Arc’s CA domain to the Arc 5’ UTR mRNA sequence and how this selective interaction can initiate Arc multimerization for capsomere assembly.

Cyanobacteria are the only known prokaryotes that can photosynthesize. Due to their unique ability, they are important entities in the global carbon and nitrogen cycles. However, two factors can turn cyanobacteria into a nuisance. In general, eutrophication is the process that supplies external nutrients that are needed for cyanobacteria blooms into bodies of water from the surrounding environment. Climate warming intensifies this process by producing carbon dioxide, methane, and nitrous oxide. With cyanobacteria blooms occurring worldwide, they are becoming a bigger threat to human health, and there has been increased interest in detecting cyanobacteria and their toxic byproducts. For instance, cytotoxins produced - such as microcystin-LR (MC-LR) - can be detected with an immunoassay using an antibody and total internal reflection ellipsometry (TIRE), or enzyme-linked immunosorbent assay. Human health effects due to MC-LR exposure include several severe symptoms: vomiting, flu-like symptoms, abdominal pains, kidney and liver damage. Thus, there is a need to detect MC-LR before the toxin levels make cyanobacteria a health concern. To address this need, we hypothesize that we can detect MC-LR using a fully customized aptamer and a novel on-site water system biosensor. Originally, we had focused on the cyanobacteria itself, specifically the A. flos aquae strain, as the target of detection. However, we changed our focus to MC-LR since the World Health Organization (WHO) considers the concentration of MC-LR when determining if a body of water is contaminated or not. The definition of toxic drinking water as stated by WHO is 1 microgram of MC-LR per liter of water.

Advances in life sciences in recent decades have revolutionized our understandingof biochemical and biophysical interactions associated with diseases and disorders of the human body. This newly acquired knowledge has fueled intense interest in a range of biotechnological strategies that can improve health outcomes, spanning from biosensors to drug development to tissue engineering. There is thus an increasing need for even better understanding of biomolecular interactions at nanoscale to explore new medical frontiers, and for powerful analytical tools of increasing complexity and diversity in multiplexed bioanalysis. Surface plasmon resonance (SPR) is a core optical spectroscopic principle in the bioanalytical sphere, and its label-free methodology for bioassays has been broadly applied in drug discovery, medical diagnosis, and environmental monitoring. Advances in materials sciences, however, have provided new opportunities for re-invention of the technique and expansion of the range of analyses by SPR. The aim of this dissertation is to develop and improve the fundamental technological diversity of SPR based techniques for enhanced biosensing applications.The main strategy for the development takes the form of integrating novelmethodologies and new materials to the SPR bioanalytical workflows. First, an orthogonal analytical platform was developed by combining SPR/SPR imaging with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). A multistep functionalized plasmonic microarray was developed into a new mode (SPR-MALDI) for the sensitive detection of bacterial toxin proteins in complex environmental matrices. The combination of the techniques allowed for both quantitative determination and unambiguous qualitative identification of biological identity of the target. Second, SPR techniques were integrated with three-dimensional (3D) printing for enhancing analytical performance. A novel hybrid 3D printing and PDMS molding process was developed that overcomes fundamental resolution limits of the 3D printed optical components for spectroscopy. Prisms of multiple geometries were fabricated that demonstrated surface roughness comparable to commercial, glass-based components, providing economical alternative while yielding high sensitivity towards SPR biosensing of protein targets. Finally, we have developed a high performing SPR platform based on a more fundamental shift, switching the plasmonic material from gold to aluminum. Al thin films under Kretschmann configuration demonstrated a 60 % higher optical sensitivity in imaging mode and reduced surface fouling by 75 %. They proved excellent substrates for array-based chemical surface modifications by ionic polymers that were further employed for successful analysis of urine-based chemokine biomarkers. The work presented here should pave the way for more complex modalities in developing the next generation of biotechnology.

Understanding biomolecular interactions and cellular activities based on these interactions are important research topics in bioanalytical chemistry that can have a large impact on key fields such as drug discovery, biomedical sciences, and environmental monitoring. High performing biosensors are an essential part for this task. Effective sensing platforms with improved analytical performance and reduced cost are actively sought after and remain a major area of intense research endeavors. The goal of this thesis is to develop novel biosensing systems enabled by 3D printing technology and nanoscience for probing biochemical interactions and achieving cell lipidomic analysis.Surface plasmon resonance (SPR) biosensors have been wildly applied for biomolecular assays in a label-free fashion. The sensitivity of SPR sensors, however, still needs signal enhancement when dealing with trace amounts of analytes. Chapter 2 describes the fabrication and application of gold nanoparticles-coupled POPC liposomes nanoclusters for signal amplification for SPR sensors and the study of protein-membrane recognition on a biomimetic interface. By combining the large mass of liposomes and plasmonic coupling of the noble metal nanoparticles, the highly stable POPC-gold nanoparticles showed a large amplification effect and a 0.1 ng/mL LOD was achieved for cholera toxin detection. 3D printing technology has greatly impacted the bioanalytical fields by providing the fabrication capability with almost no geometric restriction, rapid prototyping, and low cost. Chapter 3 describes the design and fabrication of a Dove prism by 3D printing with transverse micropatterns for multiplexed sensing with SPR imaging. The sensitivity of the system was evaluated by bulk refractive index change and protein binding assays; both showed similar performance as compared to the SPR configuration using high end glass optics. 3D printing technology has been further applied to fabricating microfluidic chips for cell analysis. Chapter 4 demonstrates the design and fabrication of a 3D printed microfluidic device for on-chip cell lysis, lipid extraction and phase separation for lipidomic study of algae C. reinhardtii cells by the microchip enhanced MALDI-MS. Compared with conventional bulk methods, extraction by the microfluidic device showed higher efficiency and cell lysis capability.

Abstract: Over the past decades, surface plasmon resonance (SPR) technology has gained popularity as a powerful analytical technique for the study of biomolecular interactions and medical diagnostics. This is made possible thanks to the well-established gold-thiolate chemistry. However, the limited sensitivity and long-term stability of gold substrates and alkanethiol self-assembled monolayers motivate us to explore lamellar carbon-on-metal structures as SPR substrates and the spontaneous adsorption of aryldiazonium salts as surface functionalization strategies. Our electron-beam evaporation process allows very thin, defect-free, well-adhered carbon films to be deposited on metal substrates, leading to minimal sensitivity loss for carbon-on-gold films and enhanced sensitivity for carbon-on-silver films in SPR measurements compared with previous reports. With further optimization of the deposition process and SPR instrumental setup, we believe that the sensitivity of carbon-on-silver films can be increased even more. Spontaneous adsorption of aryldiazonium salts provides a fast and simple strategy to pattern surfaces selectively with stable monolayers of chemical linkers, which are viable for the covalent attachment of biomolecules on SPR substrates. This surface chemistry, together with carbon-on-gold films, were utilized to develop a proof-of-concept immunoassay, showing improved performance compared with the physical adsorption of biomolecules. This thesis demonstrates that a combination of carbon-on-metal films and spontaneously grafted diazonium layers can be a potential alternative to Au-thiolate SAMs for SPR sensing, thereby improving the performance of SPR-based biosensors. What is more, carbon-on-metal substrates also have potential as a versatile analytical platform that are compatible with not only SPR but also electrochemistry and SERS techniques, opening a pathway for new, exciting nanostructures and sensing schemes to be developed with analysis power never seen before.

Nature is a constant inspiration for us. The mechanisms on which life is based are, at the same time, incredibly complex and surprisingly harmonious; individual macromolecules interact in a seemingly chaotic dance, but which has an order and a melody. Amyloid fibrils are a class of bio-macromolecules that is being increasingly brought under the spotlight. In vivo, amyloids can be both pathological and functional. In fact, they are responsible of severely debilitating diseases, such as Alzheimer’s and Parkinson’s, but they also assume pivotal roles in key biological functions. In vitro, amyloid fibrils synthesized from food sources are more and more used as building blocks of innovative materials, the applications of which range from filtration of water pollutants to drug delivery. Consequently, extensive studies on how amyloid fibrils interact and how they form gels are pivotal, both for finding novel therapeutic approaches for the above-mentioned diseases, and for optimizing the synthesis of materials that meet human needs, while preserving the resilience of our Planet. Fibronectin is another important biomolecule, which is largely present in the extra-cellular matrix. The heparin-mediated conformational dance of fibronectin tunes its interactions with other key molecules, such as growth factors and, among these, the Vascular Endothelial Growth Factor (VEGF, the role of which is crucial for angiogenesis). A deeper understanding of how fibronectin and VEGF interact would lead to the possibility of synthesizing more effective drugs, the target of which is angiogenesis in pathological conditions (like cancer). This doctoral thesis contains studies on both the biomolecules mentioned above. Regarding amyloid fibrils, the aim of the studies we present is to characterize amyloid fibril gels (both over the course of their formation and structurally, when already formed), and to strengthen them through inclusion of polysaccharides. More in details, we show that in amyloid gels, the preparation of which is based on the passage of ions through a semipermeable membrane, the dynamic of the ions is non-diffusive but, instead, advective. This suggests the presence of a force that acts on the ions, that we hypothesize being arising from the Donnan effect. We also show that the ionic strength of the gels has a key role in tuning the entity and the frequency of restructuring phenomena, the role of which is to release the stresses accumulated by the fibrils over the gelation process. From a structural characterisation perspective, we show that the mesh size of amyloid networks can be measured through Dynamic Light Scattering (DLS) experiments, combining the theory of scattering from semi-flexible polymers together with a rigorous approach that takes the nonergodicity of the studied materials into account. Finally, with a more applied focus, we show an approach to strengthen amyloid fibril hydrogels and aerogels through inclusion of polysaccharides. The sugar chains improve the compression resistance of the materials, without interfering with the surface properties of the amyloid fibrils. In the case of fibronectin, we show how this molecule is able to bind not only with VEGF, but also with an important receptor of this growth factor (VEGFR2). More in detail, heparin has an important role in extending fibronectin molecules, exposing binding sites that can interact with both VEGF and VEGFR2. The interactions are favoured at acidic pH, due to modifications in the protonation state of the binding sites of the ligands: this piece of information deepens the understanding of how hypoxia and low extracellular pH are related to angiogenesis. Moreover, thanks to Surface Plasmon Resonance studies and their modeling, we show that not all the fibronectin molecules interact equally with VEGF and VEGFR2, and can instead be divided into two classes, with different kinetics and affinities. To resume, the presented studies broaden the actual knowledge on amyloid fibrils and fibronectin, and pave the way for synthesizing new functional materials and for facing severe diseases with novel therapeutic approaches.

Interfaces are ubiquitous in our daily life. A good understanding of the interfacial properties between different materials, or a single material in different physical states is of critical importance for us to explore the current world and bring benefits to mankind. In this work, interfacial behavior was investigated with the help of surface analysis techniques, such as quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR) and atomic force microscopy (AFM), in order to gain better understanding on biofuel conversion, gene/drug delivery, and chemical fixation of CO2. Biomimetic chelator-mediated Fenton (CMF) non-enzymatic degradations on cellulose and chitin thin films was studied by liquid-phase QCM-D and AFM. QCM-D is a powerful tool to monitor the kinetics of hydrolysis of regenerated cellulose and chitin model surfaces. Results from QCM-D and AFM showed that the majority of the biomass of the two model surfaces can be hydrolyzed by the CMF system. The initial degradation rates for both model surfaces by the CMF system are faster than that of the corresponding enzyme systems. The CMF system, which is a good non-enzymatic pretreatment agent for cellulose and chitin, may work on a wide variety of polysaccharide systems. Adsorption of cationic cellulose derivatives onto self-assembled monolayer (SAM) surfaces was investigated using liquid-phase SPR. Results from SPR showed that depending upon the cellulose derivative structure, irreversible adsorption ranging from a monolayer to ~1.6 layers of cellulose derivative were formed on the SAM-COOH surface based upon a charge neutralization mechanism. At low salt concentrations, the long-range electrostatic attraction between the cationic cellulose derivatives (6-PyrCA and 6-MeIMCA) and the SAM surfaces facilitates the formation of a 2-dimensional monolayer. While, for TMACE, the energy gained through the hydrophobic interaction between adjacent long polyelectrolyte branches may afford the electrostatic repulsion and chain entropy penalties, resulting in the formation of 3-dimensional adsorbed polyelectrolyte layers. Adsorption of 1,2-epoxybutane gas molecules onto/into VPI-100 metal–organic frameworks (MOFs) was studied by gas-phase QCM-D experiments. Results from QCM-D demonstrated that VPI-100 (Ni) MOFs have higher irreversible adsorption per unit cell (θ) and faster diffusion coefficients (D) than VPI-100 (Cu) MOFs. The presence of bound counter-balancing ions on the metallo-cyclam core was attributed as the cause of the higher θ and faster D through the Ni analogue, which suggests the MOF-epoxide interaction occurs at the metallo-cyclam. This study shed light upon tuning MOF structures for better CO2 sorption and epoxide activation to gain higher catalytic efficiency. Finally, in operando high energy X-ray diffraction (HEXRD) was used to monitor the phase transition of the NaxNi1/3Co1/3Mn1/3O2 cathode material during the sintering process. The first charge/discharge cycle of the NaxNi1/3Co1/3Mn1/3O2 cathode materials in different phases were also studied by in operando HEXRD. It was found that the intergrowth P2/O1/O3 cathode (NCM-Q cathode) can inhibit the irreversible P2–O2 phase transition and simultaneously improve the structural stability of the O3 and O1 phases during cycling. The NCM-Q cathode with triple-phase integration demonstrates highly reversible phase evolution during high voltage cycling, possibly leading to a highly reversible capacity and good cycle stability.

H2 has long been known to be one of the highest energy density fuels. Fuel cell technology has been actively pursued as an environmentally friendly power source for automobiles. The promise of the technology is hindered by the lacking of a sustainable way of producing H2. Solar water-splitting via photo-electrochemical cells (PEC) is the most promising approach which converts the sustainable solar energy to the chemical energy store inside the H2 bonding. The materials that can be used in a PEC cell must fulfil a variety of thermodynamic and kinetic requirements to ensure good efficiency and durability. Single-layer MoS2 possess the corrected energy bandgap of 1.9 eV which allows for sufficient over-potential while still being capable of absorbing the majority of the solar spectrum. However, the limited optical absorbance from single-layer MoS2 prevent it from widely used. This shortcoming of the single-layer thickness of MoS2 is overcome by superimposing the MoS2 with plasmonic surface that serves to amplify the enhanced electromagnetic field where the MoS2 is located, allowing single-layer MoS2 to efficiently absorb Sun light thus producing H2. In the first part of dissertation, a novel two-step chemical vapor deposition method is developed to consistently grow high coverage and exclusive single-layer MoS2. Up to 90% surface coverage and single-layer MoS2 is successfully fabricated. This is a crucial step to conduct the follow-up experiments.In the second part of dissertation, A facile one-pot synthetic approach for synthesis hollow Au nanoframes structure is reported for the first time. A growth mechanism has been revealed that involves a synergistic function of Ag and Br ions. The presence of Ag+ lead to observed self-limiting of Au film thickness whereas Au {111} facets are preferentially attacked by the presence of Br- in the reaction ambient. Combined simulation and experimental studies show strong plasmonic effect that the hybrid platform made of graphene/Au nanoframes is capable of detecting analytes at concentration levels down to 10-9 M by using the surface-enhanced Raman spectroscopy (SERS) technique. In the last part of the dissertation, the plasmonic effect generated from nano-structured metal surfaces is introduced to offset the small thickness from single-layer MoS2 and improve the overall absorption. It is demonstrated for the first time that using single-layer MoS2 as a well-defined nanospacer between Au-nanoparticles and Au-film (gap plasmon system). The field enhancement is known to be inversely proportional to this gap thickness. Hence reducing the gap thickness is important to achieve the highest possible field enhancement. �In this work, it is demonstrated for the first time that using single-layer MoS2 as a well-defined nanospacer between Au-nanoparticles (AuNPs) and Au-film, which could offer an extremely high localized electric field enhancement within the gap. The MoS2 Raman intensity with the Surface Enhanced Raman Scattering (SERS) enhancement factor (EF) up to 5x106 is obtained from the MoS2-Au gap plasmon system. A 5-fold increase in the photocurrent is obtained from the MoS�2-Au gap plasmon as the working electrode compared to that from bare MoS2 prepared under the same condition. Compared with individual metal nanoparticles commonly used to enhance thin-film photocatalytic process, gap-plasmon could theoretically produce 8 orders of magnitude higher SERS EF and precise control the hot spot location to superimpose where ultrathin materials locate thus using the higher incident energy available.

Gold nanoparticles (AuNPs) have been brought to the forefront of various applications, ranging from theranostics, to organic photovoltaics, to biosensing owing to their localized surface plasmon resonance (LSPR) property. However, this property needs to be improved in order to allow for high sensitivity and quantitative detection of biomolecules. Hybrids of AuNPs with low-dielectric cellulose nanocrystal (CNCs) would yield enhancement of the LSPR property, which is driven by the confinement of electron oscillation at their interfaces. This study proposed a seed-mediated growth method to synthesize hybrids of CNCs-AuNPs. Sulfate groups on the surface of CNCs served as the sites for the growth of AuNPs. Optimization through the number, size, and distances of AuNPs on the surface of CNCs was achieved by modulating different variables such as concentrations of reducing agent and CNCs, time, temperature, and surface charge of CNCs. The results via absorbance spectrophotometry, dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) demonstrated that AuNPs grew on the surface of sulfonated CNCs, leading to enhancing their LSPR. Optimization and tuning the optical response of AuNPs on the surface of CNCs was accomplished in this study, which could significantly advance the technology of biosensors in the future. This study could be expanded by the utilization of CNC as a template for the growth of other nanoparticles for a variety of applications, ranging from biomedicine to optoelectronics.

Disulfide lipoic acid was used to synthesize new aqueous soluble gold nanomaterials. The first one is an ultra-small plasmonic nanoparticle displaying a weak plasmonic band at 520 nm. At the transition zone between larger metallic nanoparticles and smaller non-metallic nanoclusters, intriguing electrochemical and optical features were observed. The other cluster is a new molecular-like nanocluster with unique optical features. Distinct UV-visible absorption bands were observed corresponding to discrete energy states/orbitals along with a weak photoluminescence if ultracentrifuge purification is adopted. Dialysis purification yielded a tenfold increase in photoluminescence while the absorption bands diminish. This transition is attributed to the gradual oxidation of some of the sulfur atoms at the core-ligand interface. Annealing with a known amount of excess thiol is shown to expedite and better control the transitions observed through the synthesis and purification along with yielding an enhancement of the electrochemiluminescence by more than ten folds.

Non-equilibrium systems have no set method of analysis, and a wide array of dynamics can be present in such systems. In this work we present three very different non-equilibrium models, inspired by biological systems and phenomena, that we analyze through computational means to showcase both the range of dynamics encompassed by these systems, as well as various techniques used to analyze them. The first system we model is a surface plasmon resonance (SPR) cell, a device used to determine the binding rates between various species of chemicals. We simulate the SPR cell and compare these computational results with a mean-field approximation, and find that such a simplification fails for a wide range of reaction rates that have been observed between different species of chemicals. Specifically, the mean-field approximation places limits on the possible resolution of the measured rates, and such an analysis fails to capture very fast dynamics between chemicals. The second system we analyzed is an avalanching neural network that models cascading neural activity seen in monkeys, rats, and humans. We used a model devised by Lombardi, Herrmann, de Arcangelis et al. to simulate this system and characterized its behavior as the fraction of inhibitory neurons was changed. At low fractions of inhibitory neurons we observed epileptic-like behavior in the system, as well as extended tails in the avalanche strength and duration distributions, which dominate the system in this regime. We also observed how the connectivity of these networks evolved under the effects of different inhibitory fractions, and found the high fractions of inhibitory neurons cause networks to evolve more sparsely, while networks with low fractions maintain their initial connectivity. We demonstrated two strategies to control the extreme avalanches present at low inhibitory fractions through either the random or targeted disabling of neurons. The final system we present is a sparsely encoding convolutional neural network, a computational system inspired by the human visual cortex that has been engineered to reconstruct images inputted into the network using a series of "patterns" learned from previous images as basis elements. The network attempts to do so "sparsely," so that the fewest number of neurons are used. Such systems are often used for denoising tasks, where noisy or fragmented images are reconstructed. We observed a minimum in this denoising error as the fraction of active neurons was varied, and observed the depth and location of this minimum to obey finite-size scaling laws that suggest the system is undergoing a second-order phase transition. We can use these finite-size scaling relations to further optimize this system by tuning it to the critical point for any given system size.

Abstract: Surface plasmon resonance (SPR) has emerged as a powerful tool in sensing and biosensing for decades due to its label free and real-time data collection characteristics. In the first part of this thesis, a custom-built high performance SPR spectrometer with broad range of scanning angle, high resolution of 0.001° and multi operation mode is described in Chapter 2. The second part of the thesis (Chapters 3-5) focuses on development of a novel SPR sensor substrate that is able to detect small molecules (< 400 Da) in low concentration with high sensitivity. Stimuli-responsive polymers, especially poly(N-isopropylacrylamide) (pNIPAm)-based microgels, were used in the assay development for SPR signal enhancement (Chapters 3-4). In addition, a cost effective method was developed to fabricate hexagonal close packed Au nano arrays by directly painting the Au@pNIPAm hybridized core shell particles on the substrate and plasma etching after deposition; this can potentially be used in SPR sensor substrate and surface-enhanced Raman spectroscopy. Due to the thermoresponsivity of the pNIPAm shell, the interparticle Au nanoarray distance can be dynamically tuned and controlled by varying the painting temperature. In Chapter 6, a temperature-light dual responsive photonic device composed of Au@pNIPAm core-shell microgels is reported and its optical properties were investigated under the stimuli of temperature and light. In addition, the dependence of AuNPs distribution in microgel composites on light responsive performance was compared in terms of optical heterogeneity and responsive kinetics. In Chapter 7, a pNIPAm-brush based optical device with multi-responsiveness was reported, which can potentially be used for sensing and biosensing, drug delivery or other applications that require light manipulation and wavelength filtration.

Abstract: Small-molecule metabolites (MW < 1,000 Da) are now emerging as an important type of biomarkers, thanks to the rapid development of systems biology such as metabolomics. Compared to nucleic acids and protein, metabolite is closer to the phenotype. Rapid detection and quantification of metabolites can potentially provide an effective way to link the metabolite profile to disease state. A sensor based approach for the measurement of metabolites can fulfill the simplicity and portability required for widespread use. Surface plasmon resonance (SPR) combined with molecular recognition elements to deliver high specificity is a sensing platform that has been widely applied for a large range of biomolecules. However, direct detection of small molecules such as metabolites with SPR challenges the refractive-index based detection mechanism due to their low molecular weights. To this regard, sensing strategies that can tackle this challenge are highly demanded. In this thesis, I successfully developed a series of indirect sensing strategies (competition, inhibition, displacement), some of which are using either small molecule or aptamer functionalized gold nanoparticle labels, to detect a few single metabolite targets. All the strategies have shown great analytical performance with good sensitivity and specificity. In each assay, either periplasmic binding protein, or antibody, or DNA aptamer, was served as the recognition element, which accounts for the high specificity observed. More importantly, the multiplexed detection of a panel of metabolites through a single SPR measurement was accomplished for the first time, based on the small molecule (micro)arrays patterned on our homemade SPR imaging chip. Compared to SPR sensing of nucleic acids and proteins, the power of this technique was underestimated in metabolites sensing. I hope this thesis can provide some insights to the SPR community and inspire more work to be done in terms of small molecule sensing, thus having more impact in the fields of medical diagnostics, therapeutic drug discovery, food safety control, and environmental monitoring.

On the nanoscale, size and structure are powerful dictators of optical and electronic response. As such, they may be rationally manipulated in order to obtain desired properties for specific applications. The dissertation projects herein are aimed toward understanding how structure affects the photophysical properties of three nanoparticle systems: hollow gold nanospheres (HGNs), doped α-Fe2O3 nanostructures, and PbS/CdS core/shell quantum dots (QDs). The dissertation is divided into two parts: Part I: The Highly Tunable Hollow Gold Nanosphere: Synthesis, Size, and Surface Morphology and Part II: Ultrafast Charge Carrier Dynamics of Hematite Nanostructures and PbS/CdS Quantum DotsPart I focuses on synthetic control and characterization of HGNs, solvent-filled plasmonic shells of gold ranging from 20-200 nm in diameter. HGNs have shown promising performance in drug loading, targeted delivery, surface-enhanced Raman scattering, and photothermal therapy. Their optical properties are very sensitive to their aspect ratio, the ratio of diameter to shell thickness. As such, a well-controlled synthesis is highly desired. In Chapter 1, the HGN synthesis was updated to enable simultaneous control of both diameter and SPR while maintaining monodispersity and uniformity of the resultant shells. This was possible through a detailed and systematic investigation of the synthesis of the sacrificial cobalt-based scaffolds onto which HGNs are formed through galvanic exchange.In Chapter 2, additional synthetic adjustments were introduced to systematically control the HGN surface morphology from smooth to very bumpy. Rugose structures are perhaps the most versatile nanoparticles, with an increased density of active sites for catalysis as well as local electric field enhancement around the surface features for sensing and detection. As hollow particles have displayed enhanced plasmonic performance in comparison with their solid counterparts for a number of applications, the combination of hollow cores and rugose surfaces is highly attractive. One of the most attractive applications of HGNs is plasmonic photothermal therapy (PTT). In this application, plasmonic nanoparticles are targeted to cancer cells where they convert incident light to heat, raising the temperature of their environment above the point of cell viability. A systematic comparison of HGNs with different surface morphologies revealed that bumpy HGNs retain the excellent photothermal conversion efficiency (PCE) of their smooth counterparts. Next, in Chapter 3, PCE was investigated for HGNs of different diameters, theoretically and experimentally. The findings revealed that 50 nm HGNs generate ~2 times the heat per µg gold as their 70 nm counterparts and ~1.5 times the heat per µg gold as their 30 nm counterparts. In vitro HGN-mediated PTT of oral squamous cell carcinoma was also carried out. Ongoing efforts are needed to assess the PCE and potential size dependence of HGNs in vitro and in vivo.In Part II, transient absorption spectroscopy (TAS) is used to probe the charge carrier dynamics in α-Fe2O3 (hematite) and PbS/CdS nanostructures. In both systems, structural modification has been crucial for obtaining enhanced photophysical performance. Hematite is considered to be an especially promising material for solar water splitting due to its chemical stability, abundance, and non-toxicity. Additionally, its band gap of approximately 2.0–2.2 eV facilitates the absorption of about 40% of incident solar light. However, it suffers from a number of limitations which hinder its practical implementation as a photoanode material. Chapter 4 discusses these limitations and structural approaches to overcome them, including nanostructuring and doping to facilitate charge transfer and improve PEC performance. Charge carrier recombination dynamics are investigated before and after doping to aid understanding of the mechanism of performance enhancement in Zr and Ti-doped hematite films. Although nanostructuring and doping are beneficial, performance gains have been modest and a review of the current approaches for the rational design of hematite heterostructures is provided in Chapter 5. In these reports, TAS has been employed as a useful tool to gain deeper insight into the mechanisms of photogenerated electron-hole recombination and their relation to PEC performance.Finally, in Chapter 6, the passivation of PbS QDs with a CdS shell, and the dependence of the passivation on particle size, is investigated. Semiconductor QDs like PbS are highly attractive components in solar cells, sensing, and detection due to their size-dependent optical and electronic properties. Broad absorption, narrow photoluminescence (PL), and high PL quantum yield arise from quantum confinement. The formation of a thin shell atop the QD core has become a common method for passivating the surface bonds and thereby enhancing and stabilizing resultant PL. In this work, it was found that surface passivation of PbS with a CdS shell is core-size-dependent; ultrasmall PbS QDs did not benefit from CdS passivation like their larger counterparts, but instead experienced a decrease in PL. Coupling TAS with steady state PL and QY measurements enabled a comprehensive understanding of the radiative and nonradiative relaxation pathways of the PbS/CdS nanosystem. Insights into the size-dependent variation in trap state density and relaxation pathways may serve as a guide for future structural modification.

Nanotechnology has advanced to the point that nanoparticles can now be fabricated in a broad variety of shapes from a wide range of materials, each with their own properties and uses. As the list of manufacturable particles continues to grow, a new frontier presents itself: assembling these existing nanoparticles into more complicated nanoscale structures. The primary objective of this thesis is to demonstrate and characterize one such method of nanoscale construction, the plasmonically directed self-assembly of gold nanospheres onto both silver nanospheroids and gold nanorods. At the heart of this research is a the use of a photocleavable ligand (1-(6-Nitrobenzo[d][1,3]dioxol-5-yl)ethyl(4-(1,2-Dithiolan-3-yl)butyl) carbamate), which is capable of forming a photoreactive self-assembly monolayer (SAM) on gold and silver surfaces. After photoactivation, this SAM becomes positively charged at low pH, allowing it to electrostatically bind with negatively charged gold nanospheres (or other negatively charged nanoparticles). In this thesis, I describe both a secondary photoreaction that this ligand is capable post-photocleavage, which removes the ligand's ability to bind to negatively charged gold nanospheres, allowing for, among other assembly methods, reverse photopatterning. I further show that this photocleavable ligand can be used in conjunction with gold nanospheres to create aligned, metal structures on silver nanospheroid surface by exposure to linearly polarized UV light. Similarly, I also demonstrate how the ligand can be used to preferentially bind gold nanospheres to the ends of gold nanorods with the use of ultrafast femtosecond pulsed 750 nm laser light, making use of multi-photon absorption. Both methods of self-assembly, as well as the secondary photoreaction, are dependent on the plasmonics of the metal nanoparticles. This thesis also goes into the backgrounds of plasmonics, plasmonically mediated catalysis, self-assembly, and photocleavable chemicals.

Biomarkers are any components in the human body in the form of biomolecules, cells, tissues, or fluids that can be measured and evaluated to facilitate the indication of healthy processes, pathological processes, or pharmacological responses to a therapeutic intervention. Numerous ongoing research investigations in the identification of such components have offered potential means of early disease prognosis and assessment of the impact of fatigue and/or stress on human physical and cognitive performance. Currently, studies on human performance biomarkers are some of the highly investigated areas in this field. These include, but are not limited to lactate, cortisol, and orexin A for the quantification of physical and cognitive state. It is preferred to monitor such biomarkers unobtrusively in real time which can impact a number of professions with high-risk tasks such as surgeons, anesthesiologists, commercial pilots, and combat forces. Especially, in the military services, a device with similar capability would greatly assist in their sense-assess-augment paradigm by monitoring the performance of a soldier in the field. For the implementation of such a system, one of the most challenging aspects is bio-sample collection. Traditionally, phlebotomy is used as the gold standard for sample collection. However, it is painful, inconvenient and unsuitable for the purpose described as it can significantly limit the sampling interval. Some other noninvasive alternatives like saliva and passive sweat may be considered as possible sources. Nevertheless, they carry some shortcomings like imprecision, requirement of volume normalization, and possible need of controlled supervision for the sample collection and analysis. A technique known as reverse iontophoresis can overcome some of these issues in addition to providing a more precise sampling. It facilitates transdermal extraction of charged and neutral biomolecules up to 20 kDa in size present in the interstitial fluid by passing a low-level current between two electrodes placed over the skin. The extracts on the skin surface can then be analyzed by an on-site biosensor, or transported to a dedicated analyzer via a microfluidic pathway. It should be noted, however, the concentrations of RI sampled analytes are at significantly lower levels compared to those found in blood samples. Therefore, highly sensitive biosensors are required for the quantification of these samples. One of the widely used sensing techniques that could be potentially integrated to develop a RI-based sensing platform would be the chronoamperometric method. However, this technique may not be suitable for some larger peptides and protein-based biomarkers such as cortisol, and orexin A that require highly sensitive biosensors and potentially demand the integration of a microfluidic interface for the RI-based sample to flow over the sensor surface. Although the technologies required to develop a noninvasive biomarker sensing platform for various types of analytes are available, there is still an unmet need of a robust platform that integrates them into a single wearable device. In this dissertation, this need has been addressed by successfully developing a RI-based sensing platform that integrates amperometric and SPR-based sensors along with the necessary electronics and microfluidic interfaces. To accomplish this, commercially available application specific integrated circuits were used to design the essential electronics for a miniature RI driver module, amperometric and SPR sensor interfaces, and a microfluidic flow controller. A microfluidic interface that houses a micro electro-osmotic pump and a disposable cartridge for the sample collection and transport to a SPR-based sensor was also developed. Furthermore, a planar electrode system and enzymatic hydrogels were fabricated for chronoamperometric sensing of glucose and lactate. Ultimately, these components were integrated into a single wearable prototype.

The glycoprotein CD36 is expressed in the plasma membrane (PM) of many cell types that surround or contact arteries, including macrophages, myocytes, and endothelial cells. CD36 binds oxidized low density lipoprotein (oxLDL), which promotes atherosclerosis, and fatty acids (FA), which promotes their cellular uptake. To gain insights into the molecular mechanisms of uptake, HEK293 cells expressing CD36 were studied by cell biological and fluorescence methods. To test our hypothesis that the PM is not an impermeable barrier to FA and that FA move into cells by diffusion via their uncharged form, we first applied biophysical fluorescence spectroscopy to directly measure transmembrane FA movement and membrane fluidity. Expression of CD36 in HEK293 cells did not increase either transport across the PM or the fluidity of the PM compared to HEK293 cells without CD36; however, CD36 enhanced intracellular FA esterification. Furthermore, the widely used “inhibitors” of FA transport did not alter either the rapid FA transmembrane diffusion in HEK293 cells or diffusion in control experiments with protein-free phospholipid bilayers. To gain new insights into the physiological relevance of FA binding to CD36, we applied surface plasmon resonance (SPR) to quantify FA and oxLDL binding to the ectodomain of CD36. Structurally distinct FA [saturated, monounsaturated (cis and trans), polyunsaturated, ω-3, ω-6, and oxidized FA] were pulsed in a solubilized form (bound to methyl-β-cyclodextrin) across SPR channels, generating real-time association and dissociation binding curves. With the exception of the oxidized FA hydroxyoctadecadienoic acid (HODE), all FA tested bound to CD36 with rapid association and dissociation kinetics similar to human serum albumin. In addition, FA increased oxLDL binding to CD36. To investigate whether FA affect CD36-mediated oxLDL uptake in live cells, we monitored fluorescent oxLDL (Dii-oxLDL) uptake using confocal microscopy. Addition of exogenous FA to serum-free media enhanced dose-dependent oxLDL uptake. Exceptions were ω-3 FA, which bound to CD36, and HODE, which did not bind to CD36, demonstrating FA structure-specific effects on a major function of CD36 and a new mechanistic link between atherosclerosis and high levels of FA in obese and Type-II diabetic individuals.

In light of their escalating exposure to possible environmental toxicants, there are many biological systems that need to be evaluated in a resource and time efficient manner. Understanding how toxicants behave in relation to their physicochemical properties and within complex biological media is especially important toward developing a stronger scientific foundation of these systems so that adequate regulatory decisions may be made. While there are many emerging methods available for the detection and characterization of these chemicals, nanotechnology has presented itself as a promising alternative toward creating more efficient assays. In particular, metallic nanoparticles and thin films exhibit unique optical properties that allow for highly sensitive and multiplexed studies to be performed. These plasmonic materials often preclude the use of molecular tags and labels, enabling direct characterizations and enhancing the throughput of biomolecular studies. However, their lack of specificity toward certain targets and potential toxicity has thus far precluded their widespread use in toxicity testing. The cell membrane, a natural signal transducer, represents one of the fundamental structures for biological recognition and communication. These interfaces principally function as a selective barrier to exogenous materials, including ions, signaling molecules, growth factors, and toxins; therefore, understanding interactions at membrane interfaces is a vital step in elucidating how biological responses are effected. Supported lipid bilayers, which may easily be tailored in composition and complexity, are ideal interfaces for coupling to plasmonic assays since they may be supported in close proximity to metallic nanoparticles and thin films, where measurements are most sensitive. This research will focus on the coupling of plasmonic materials and biomimetic interfaces to increase the sensitivity, efficiency, and throughput of conventional toxicity assays. The fabrication of new plasmonic materials for membrane-based assays is presented, as well as method developments in membrane array formation and opportunities for hyphenation with complementary analytical techniques.

When illuminated with visible light, nanostructured noble metals exhibit a strongplasmon resonance at wavelength, p, that has been shown to be sensitive to its size,structure, the dielectric properties of the surrounding medium, and charge density. Thetunability of the plasmon resonance has allowed metal nanosystems to be fabricated withresonances matching the solar spectrum for us in plasmon promoted catalysis, plasmonicphotovoltaics, and surface-enhanced raman spectroscopy. Here we use UV-Visible spectroscopyto track the shifts of the plasmon resonances from an array of gold nanoparticlesburied under metal oxide layers of varying thickness when in contact with one of two bulkmetals: aluminum or silver. By assuming the array of gold nanoparticles and metal-oxidelayers to be an optically homogenous lm of core-shell particles on a substrate, we developeda Maxwell-Garnett effective medium approximation to extract reliable opticalparameters for the gold nanoparticles, yielding their charge state before and after contactwith the bulk metal.Based on the optical parameters extracted from our model, we nd the magnitude ofcharge transfer from the bulk metal to the gold nanoparticle is independent of the workfunction of the bulk metal. Furthermore, when gold is used as the bulk layer in contactwith the gold nanoparticles, we measured an appreciable amount of charge transfer to thegold nanoparticles, failing to support the well-established model for electrostatic contactelectrication. Instead, we attribute the charge transfer to the so called plasmoelectriceffect, an optically induced charge transfer mechanism, in which the gold nanoparticlemodifes its charge density to allow its resonant wavelength to match that of the incidentlight. We show, however, that in our devices the Schottky barriers between the metalsand the metal oxide layers create a rectication effect that favors electron transfer fromthe bulk metal to the nanoparticles over the reverse effect.

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.

Adaptor proteins, like Tollip and Tom1, facilitate cellular cargo sorting through their ubiquitin-binding domains. Tollip and Tom1 bind to each other through their TBD and GAT domains, respectively, whereas Tollip interacts with phosphatidylinositol-3-phosphate (PtdIns(3)P)-containing endosomal membranes. Tom1 and Tollip interaction and association with endosomes is proposed to be involved in the lysosomal degradation of polyubiquitinated cargo. Through cellular, biochemical, and biophysical techniques, we have further characterized the association of Tom1 with Tollip. Mutations in the binding interface of the Tom1 GAT and Tollip TBD complex leads to a subcellular mis-localization of both proteins, indicating that Tom1 may serve to direct Tollip to specific cellular pathways. It was determined that Tom1 inhibits the binding of Tollip to PtdIns(3)P and inhibition was reversed when mutations in the binding interface of the Tom1 GAT and Tollip TBD were present. Furthermore, it was established that, upon the binding of Tollip TBD to Tom1 GAT, ubiquitin is inhibited from binding to Tom1 GAT. It was also demonstrated that Tom1 GAT, but not Tollip TBD, can weakly bind to PtdIns(3)P. Consequently, we propose that association of Tom1 may serve to direct Tollip for involvement in specific cell signaling pathways. Gaining insight into the function of Tom1 and Tollip may lead to their use as therapeutic targets for increasing the efficiency of cargo trafficking and also for patients recovering from various cardiac injuries.

Abstract: Our primary goal of this study was to obtain a better understanding for the carbohydrates binding interactions with human intelectin (hIntL). Studying the binding specificity of hIntL could help in discovering its natural substrate and might give us a clue about its role in the innate immune response as well as its pathophysiological action in various diseases. In this project, recombinant expression of intelectins was successfully done using HeLa cells and H5V cells. Yeast (Pachia pastoris) did not proove to be a promising expression system for hIntL. Using chemical synthesis, compounds (3.1–3.9) were obtained to be screened as ligands for hIntL. Herein, we used surface plasmon resonance (SPR) as our analytical tool to study hIntL binding specificity towards various synthesized Galf-containing oligosaccharides. Staudinger ligation chemistry as well as amine coupling were used to immobilize the carbohydrate epitopes to the SPR biosensor surface. Both hIntL-1 and hIntL-2 were screened against the derivatized surface.

We have designed and tested a new plasmonic biosensor, featuring a centered nanohole in the base of a recessed metallic nanocup. This configuration enables us to perform independent plasmon-resonance-enhanced single-nanohole transmission spectroscopy on femtoliter volumes of solution. In this thesis we will demonstrate the fabrication, characterization, and application of these novel cup resonator plasmonic biosensors. Utilizing plasmonic confinement to enhance and modulate transmission through a nanohole aperture, the resulting transmission spectra can be used to determine changes in the material properties of a dielectric material located inside the sensing volume of the cup. We have determined, through measurements and simulations, the physical mechanisms causing transmission modulation through the structure. Utilizing this information, we have constructed predictive behavior models for the design and customization of these devices for specific purposes. We show that these structures are responsive to refractive index changes in their surroundings, and propose some possible application of these resonators in biological sensing roles which take advantage of their unique geometry.

Periodic metal nanoparticle arrays possess electromagnetic properties useful for metamaterials, light trapping, and molecular sensing, such as surface enhanced Raman spectroscopy (SERS). The localized surface plasmon resonance (LSPR) of these arrays depends on the material, periodicity, size, and spacing of nanoparticles, all features typically best controlled by traditional top down nanofabrication techniques, such as electron beam lithography. However, such fabrication methods are not realizable commercially. Bottom-up methods are increasingly being explored for alternative nanofabrication techniques in order to achieve high throughput and inexpensive fabrication. I present here multiple unique techniques for facile, inexpensive fabrication of nanostructures for biosensing. In the first technique, I fabricate 20 nm Au nanoparticle nanocluster assemblies onto a chemically functionalized diblock copolymer film, through electrophoresis and convective assembly, attaining interparticle spacings of < 1 nm. Norepinephrine is detected at 200 parts per billion using SERS, due to the high enhancement fields of the cluster. This is a cheap, high through put, and commercially accessible process. The second technique uses thermally responsive, pre stressed polyolefin (PO) film to reduce bow tie arrays by 50% in triangle area and up to 77% in tip to tip spacing, by heating in a convection oven. Reduction of the arrays shows tunability (100 nm blue shift) of the plasmon resonance, as well as potential for facile generation of hot spots for SERS. I also present work on tunable nanopillars using the polyolefin film, and on SiC nanopillar arrays, for the excitation of localized surface phonon polariton modes (SPhP), as potential low loss alternatives to plasmonics. FTIR reflectance spectroscopy measurements show the nanopillar arrays to confine infrared light with the emergence of localized SPhP modes, which exhibit dependence on pillar diameter and inter pillar spacing. Extreme light confinements and low losses are attained, with Q factors higher than the best plasmonic systems. Finally, I present work using electron beam lithography in tandem with the poyolefin film to achieve advanced plasmonic structures.

With relevance spanning from disease diagnostics such as immunohistochemistry to immunoassays and therapeutics, antibody reagents play a critical role in the life sciences, clinical chemistry, and clinical medicine. Approaches such as immunohistochemistry (IHC), and immunoassays, all work by exploiting the principle that antibodies bind specifically to antigens of interest. IHC uses antibodies to detect antigens on cancerous tissue, and immunoassays use antibodies to identify biomarkers both for a myriad of disease diagnostics for cancer and infections diseases. Currently, antibody-based proteomic approaches such as immunohistochemistry and technologies that serve to diagnose and advance therapeutics are severely limited due to non-specific antibody binding, low specificity, and reproducibility issues. Selecting antibodies based on their antigen binding kinetic properties, such as their association and dissociation rate constants, kon and koff, can provide a quantitative metric that can further optimize and validate immunoreagent selection. These rate constants quantify the ability for an antibody to associate (bind) or dissociate (unbind) to a target analyte and determines inherent binding strength. Therefore a metric such as this has the power to eliminate problems seen by clinicians, researchers, and drug developers alike in regards to false positive, false negatives, and problems with reproducibility seen in antibody-based approaches and inform assay design. Consequently, scalable and efficient analytical tools for informed selection of reliable antibody reagents would have wide impact. In this work, I have developed a highly scalable, rapid, microfluidic screening assay, that is able to assess important but difficult to characterize interaction kinetics for antibodies and protein-protein interactions. This work includes a novel screening assay for quantitative characterization of binding kinetics for the development of new biomarker discovery, disease diagnostics, and novel therapies and advances antibody-based proteomics.

Plant cell wall polysaccharides are abundant natural polymers making them potential sources for sustainable and biodegradable materials. Interfacial behavior, including adsorption and enzymatic degradation, of several plant cell wall polysaccharides and their derivatives were studied with a quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR) and atomic force microscopy (AFM). Xyloglucan adsorption isotherms were obtained to probe how cellulose-hemicellulose interactions were affected by the type of cellulose substrate and molar mass of xyloglucan. Xyloglucan as small as a heptasaccharide still adsorbed irreversibly onto cellulose. Carboxymethyl cellulose (CMC) adsorption onto cellulose and viscoelastic properties and water contents of the adsorbed CMC layers were obtained from a combination of QCM-D and SPR data. The CMC samples formed hydrated and viscoelastic layers compared to the relatively rigid xyloglucan layer. Pectin model surfaces were prepared by pectin adsorption from citric phosphate buffer onto gold substrates. These pectin model surfaces were used for subsequent interaction studies with xyloglucan and enzymatic degradation behavior. There is a strong correlation between the degree of esterification (DE) and film resistance to degradation with the high DE being the most susceptible to degradation. The adsorption of two mixed linkage glucans (MLG), barley and lichen MLG, onto regenerated cellulose (RC) surfaces in the absence and presence of other matrix polysaccharides was studied. Viscoelastic properties of the resulting layer were compared as a function of the proprotion of '-(1''3) linkages with lichen MLG forming softer gel-like layers on RC. The lichen MLG layers were further used for enzymatic degradation studies with respect to enzyme concentration, temperature, pH and ionic strength. These studies show that polymer adsorption is a promising strategy to modify material surfaces and provides fundamental understanding of interactions and biodegradation of cell wall polysaccharides at solid/liquid interfaces.

Natural polymers from renewable resources have attracted increasing interest as candidates for renewable energy and functional materials. In this work, the interactions between natural polymer thin films and biomacromolecules were studied via surface analysis techniques, such as a quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR) and atomic force microscopy (AFM). Chitinase activity on regenerated chitin (RChitin) films was studied by QCM-D and AFM. The optimal temperature and pH for chitinase activity on surfaces determined by QCM-D and AFM were consistent with bulk solution studies in the literature. Results from QCM-D also indicated that chitinase showed higher activity on fully acetylated chitin than highly deacetylated chitosan. Nanocrystalline chitin (Chitin NC) thin films were prepared by spincoating a nanocrystalline chitin colloidal suspension onto solid surfaces. Solvent exchange experiments via QCM-D with H2O/D2O revealed that Chitin NC films had more water than RChitin films of similar thickness. Results from QCM-D demonstrated that Chitin NC films had high bovine serum albumin loading capacity, and chitinase not only degraded, but also caused swelling of the chitin nanocrystals. Adsorption of human serum albumin (HSA) and fibrinogen (HFN) onto bare gold, regenerated cellulose (RC) and RChitin thin films was studied by SPR and QCM-D. Studies by SPR indicated that HSA and HFN formed close-packed monolayers on gold surfaces and sub-monolayers on polysaccharide surfaces, and the adsorption affinity of HSA for polysaccharide surfaces was greater than that of HFN. Results from QCM-D and SPR showed that the protein layers on polysaccharide surfaces had more associated water than proteins on gold surfaces. The dehydrogenative polymerization of monolignols catalyzed by physically immobilized horseradish peroxidase was investigated using QCM-D and AFM. Results from QCM-D and AFM showed that coniferyl and p-coumaryl alcohol underwent polymerization directly, whereas sinapyl alcohol required the addition of a nucleophile for polymerization. Studies by QCM-D and AFM also indicated that the surface-initiated polymerization was greatly affected by the support surface, monolignol concentration, hydrogen peroxide concentration and temperature. Thin films of dehydrogenative polymer (DHP), kraft (KL), organosolv (OL) and milled wood (MWL) lignins were used to study the enzymatic degradation of lignin mediated by lignin peroxidase (LiP) and manganese peroxidase (MnP). Results from QCM-D showed that the initial rates for degradation catalyzed by LiP increased in the order: KL < OL < MWL < guaiacyl DHP (G-DHP) < p-hydroxyphenyl DHP (H-DHP). In contrast, manganese peroxidase only degraded DHP films with a faster initial rate for G-DHP than H-DHP. Adsorption of hemicelluloses onto KL, OL and MWL thin films was studied by QCM-D and SPR. Results from QCM-D showed that hemicelluloses with different structures displayed very different adsorption behavior. Adsorption isotherms from QCM-D and SPR indicated that xyloglucan possessed stronger affinity for KL and OL films than MWL films. Data from QCM-D and SPR revealed that xyloglucan formed less hydrated layers on lignin surfaces compared to RC surfaces, and the adsorbed xyloglucan layers on different lignin films had similar percentages of coupled water.

The morphology and composition of a nanoparticle (NP) play a critical role in determining the NP's properties and function. To date, researchers have created a myriad of NPs of different shapes, sizes, and compositions with interesting attributes and applications ushering a revolution in medicine, electronics, microscopy, and microfluidics. In this study, gold (Au) nanosphere dimers (NSDs) have been synthesized through a novel self-assembly method. These particles were created from Au NPs mono-dispersed in aqueous solution via a process of centrifugation and capping agent replacement. Au NSDs consist of two Au NPs combined together with minimal gaps between them. Optical spectral analysis showed two wavelength bands: a wavelength band around 520 nm, which is attributed to the transverse surface plasmon resonance (SPR), and another wavelength in the near-infrared (NIR) region with a peak around 650 nm, which is attributed to the longitudinal SPR, as in the case of Au nanorods (GNRs). Synthesis of Au NSDs does not require toxic precursors, such as cetyltrimethylammonium bromide (CTAB) when making GNRs, suggesting that Au NSDs could be more clinically applicable nanotheranostic agents for molecular imaging and therapy as well as other applications such as drug delivery. It is hypothesized that G-Force polarizes the NPs, which reduces the repulsive electrical double layer, allowing attractive van der Waal's forces to dominate and bring the surfaces in contact, causing surface reconstruction at the junction. The presence of capping agents, such as citrate, on the NP surface plays a key role in the electrostatic forces that bind the two spheres into a single, stabilized dimer by preventing further NP aggregation. Other popular NPs, including platinum (Pt) and silver (Ag) NPs were also investigated and showed significant shifts in SPR, suggesting that this method can be generalized across NPs of different compositions. Although the detailed mechanisms and the applicability to other NPs with different shapes and/or compositions remain to be determined, considering its simplicity, controllability, and versatility, this G-Force driven technique could be implemented to assemble noble NP dimers with unique opto-electro-chemical properties for many applications, including optoelectronics, nanophotonics, biosensing, biosecurity, and nanomedicine.

This dissertation focuses on liquid crystals and the inherent orientational order that arises from these anisotropic materials. Quasi two-dimensional chiral patterns were scribed via Atomic Force Microscopy (AFM) with robust control onto polyimide substrate, composed of achiral materials, yielding 3D chirality that was localized to within a few nanometers of the surface. The chirality was quantified through the surface electroclinic effect. In addition, a sample with a well controlled easy axis was fabricated and a method to measure the local director orientation with 100 nm resolution using Near-Field Scanning Optical Microscopy yielded the true angular distribution function of the local order with high precision. Finally, gold nanoparticle self-assembly was studied within a cholesteric liquid crystal (CLC) film. The open sample creates a striped texture and gold nanoparticles coated with thiol are deposited within the liquid crystal matrix. Polarized Localized Surface Plasmon Resonance (LSPR) measurements show an anisotropy between light polarized with respect to the stripe orientation. Evaporating the liquid crystal revealed disordered anisometric needles measured by AFM. Generalized Mie theory calculations, in conjunction with LSPR, found a smaller nanoparticle spacing in the CLC compared to a 2D monolayer of the same nanoparticles on a rubbed substrate or 1D chains in smectic A dislocations. The closer spacing suggests trapping sites within the CLC, where nanoparticles are first localized and then aggregate under van der Waals attraction aided and enhanced by the CLC and mediated by steric forces.