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  • (2021) Arman, Seyedyousef
    Thesis
    Impedance cellular biosensors are amongst a promising type of label-free technologies in providing ongoing insights into physiological function of cells over a period ranging from several minutes to several days. However, detection of a highly specific biomolecular event using traditional impedance assays is technically challenging. The nature of impedance signal relies on the changes in the local ionic environment at the interface, providing many biochemical events at once lacking biomolecular specificity. The next decade is then likely to witness an interest in using developed impedance assays. Impedance-quartz crystal microbalance (QCM), impedance-surface plasmon resonance spectroscopy (SPR), and impedance-optical microscopy are the hybrid approaches that have been employed in the field. Integrating impedance biosensors to another sensing method, in particular new microscopies that enable identification of cellular structures and processes with a high degree of specificity, enhances the potential of traditional assays by providing additional relevant information. Herein an effective approach for accurate interpretation of impedance signal is presented. By development of optical/electrical multi-electrode chips, light was utilized for direct visualization of cell structures and processes on the surface of the microelectrode. It was essential to achieve both high throughput electrical results and high-resolution microscopy images to detect the transient changes inside the cells. Therefore, the strategy of simultaneous dual sensing was developed in three main steps. For the establishment of a reliable dual sensing readout, it was essential to use a commercial biosensing device (known as xCELLigence) in the first step. This approach enabled to compare the electrical results of developed dual biosensing device and a commercial device (as a high throughput assay for electrical measurement of subtle changes within the cell monolayer). The highly sensitive measurement of commercial device also made it possible to investigate the ongoing mechanism behind receptor/ligand activation. The signalling pathway was determined by using different pharmacological inhibitors. In a separate parallel experiment, fluorescence microscopy was used to visualise the specificity of histamine/HeLa cell interaction which was coupled to intracellular calcium rise. While it is assumed these two processes are connected, this could not be determined definitively by the sole biosensing device application. In the second step it was necessary to develop a setup with the capability of data acquisition in both the high throughput electrical setup and high-resolution fluorescence microscopy on a single platform. The first material of choice for the fabrication of this biosensor was ITO because of its electrical conductivity and optical transparency. It was shown that contribution of cells to the overall signal on the surface of ITO depends on the parameters including sensing area and width of microfingers. Furthermore, comparing the ITO results with the identical gold microelectrode revealed the ITO severely lacked sensitivity compared to gold. This was due to a better penetration of the electric field within the cell layer on the gold surface. The addition of a viewing window made a dual sensing readout possible on the gold microelectrode. Finally, the finding were used to maximize the system efficiency and precision for the detection of minute change of cells to the drug. The reduction of the microfingers down to the single cell level led to a more efficient distribution of electric field within cell monolayer. A high density of gold electrode arrays also increased the chance of individual cells blocking the current which was desirable. The added value of the developed biosensor was illustrated by studying GPCR activation in a more thorough manner using simultaneous fluorescence microscopy. The simultaneous optical/electrical experiment was performed as a powerful approach to translate specific intracellular biomolecular event contributing to the morphological changes in cell/drug interaction.

  • (2022) Gautam, Shreedhar
    Thesis
    Extracellular vesicles (EVs) are phospholipid membrane bound sacs (vesicles) produced from almost all types of cells. They are found in circulation and contain the cargo biomolecules such as nucleic acids, proteins, lipids, and amino acids. EVs are involved in trafficking these biomolecules between cells and as such have the role in physiological and pathological processes. EVs are heterogenous and revealing their heterogeneity is crucial to understand their explicit physiological and pathological roles. Current isolation techniques cannot sort EVs based on their biogenesis and provides average information instead of each EVs subtype. Thus, single EVs analysis was popular and many surface protein characterization techniques are developed. But there are no techniques available for internal cargo analysis of individual EVs. The overall aim was to develop a technique to analyse internal microRNA cargo content, if possible, for single EVs, if not from the minimum number of EVs. To achieve that goal, light activated electrochemistry, a technique where focused light beam was illuminated on the semiconductor surface and make it electrochemically active was used. The surface was protected against oxidation during electrochemical reactions by grafting self-assembled monolayer of 1,8-nonadiyne. Then, silicon-based surface was patterned with polymers, antibodies, and cells using the light patterns. As a result, the first milestone to prepare light-assisted patterned semiconductor surface was achieved for our overall aim of analysing content of individual EVs. The size range of EVs is 30 to 200 nm, still very low compared to 30 µm which is the best spatial resolution achieved for light activated electrochemistry using crystalline silicon. Thus, chapter 4 developed a technique to improve the spatial resolution of light activated electrochemistry using amorphous silicon. Amorphous silicon has short diffusion length of charge carriers compared to crystalline silicon due to the defect states in band gap called as localized states. So, charge carriers are frequently trapped in these localized states leading to 60 times improvement in spatial resolution to 500 nm. But even this spatial resolution was not enough to analyse individual EVs. So, microRNA content from pool of EVs were detected using the screen-printed electrodes in a high throughput manner instead of single EVs.

  • (2022) Qiao, Laicong
    Thesis
    There has been a rapid-growing market and academic enthusiasm for small wearable molecular diagnostic platforms driven by the growing demand for continuous monitoring of human health. Wearable devices need to be portable, stretchable, and ideally re-configurable to be able to work for different analytes. Such flexible physiological monitoring devices which are non-invasive or minimally-invasive represent the next frontier of biomedical diagnostics. They may make it possible to predict and prevent diseases or facilitate treatment by diagnosing diseases at the initial stages. However, there are many problems that restrict further applications of these devices. Firstly, there are a limited number of bio-materials which are highly flexible, biocompatible and have anti-fouling properties; such biomaterials are needed as substrates for wearable devices. Secondly, traditional biosensors used in wearable devices focus on the detection of physical signals (such as heartbeat) and small chemical molecules, e.g. Na+, K+. These are not sufficient to provide in depth health information which requires sensing of large molecules such as proteins, ideally in real time, which is currently challenging. This provides a motivation to develop highly sensitive wearable biosensors for the detection of large molecules in sweat. This thesis centres on the development of a bio-material based wearable device for continuous detection of crucial analytes in human sweat. To achieve this target, our first aim was to design a highly bio-compatible flexible material as a substrate for wearable devices. A tough and anti-fouling three-network hydrogel has been prepared by integrating a zwitterionic polymer network into a robust double-network hydrogel. Secondly, to fill the gap between technological development of continuous and non-invasive detection of different analytes in human sweat, a patterned sweat-based biosensor was created for the detection of key biomolecules. This sensor was produced by placing specific aptamers or enzymes on flexible working electrodes. In addition, nanotechnology methods have been applied to refine the bio-sensing interface to further increase the sensitivity of our sensors. Finally, a sample collection chip has been combined with our high sensitivity sensors to fabricate a wearable device for sweat bio-sensing purposes. Future research may involve integration of a commercially available wireless signal readout module with this wearable biosensing device. The outcomes of this work may provide new insights for the development of wearable devices for continuous measurement of a spectrum of analytes in sweat, as an important step towards point-of-care diagnostics

  • (2022) Li, Zihao
    Thesis
    Therapeutic proteins have long been considered difficult to mimic synthetically. While the chemistry to make very complex polymers is generally available, the tools to efficiently screen for the effect of a polymer’s structure on its biological activity have yet to be demonstrated. In this thesis, a high throughput platform was developed for the synthesis of multivalent polymer scaffolds and applied to design synthetic mimics of the chemotherapeutic protein, tumor necrosis factor related apoptosis inducing ligand (TRAIL), which triggers apoptosis by receptor clustering. The platform makes use of a simple dual-wavelength, two-step polymerise & click approach to prepare star-shaped polymer-peptide conjugates. Polymerisations were performed in open well plates at 565 nm using an oxygen tolerant porphyrin-catalysed photoinduced electron/energy transfer-reversible addition-fragmentation chain-transfer (PET-RAFT) process. Subsequent UV irradiation results in deprotection of the polymerisation friendly cyclopropenone-masked dibenzocyclooctyne (cp-DIBAC) group at the α-chain end and the click conjugation of the desired peptide. Using this approach, the valency and position of ligands on a polymer scaffold can be precisely controlled, in a high throughput manner, without purification. Leveraging this approach, libraries of star shaped polymers which present exactly one receptor binding peptide at the end of each arm were prepared and screened for their ability to bind to the target death receptor (DR5), and trigger apoptosis through receptor clustering. Structure-activity relationships generated on a colon cancer line (COLO205) led to the identification of ~ 10 kDa trivalent structures as the most promising leads, which showed IC50 values of ~ 2 µM. Elevated levels of caspase-8 were used to confirm the mechanism of cell death. The scaffold design was then iterated by introduction of hydrophobic blocks into the centre of the star polymer, which resulted in improved spatial control over peptide presentation in solution. This led to around 30-fold improvement in IC50 (75 nM). These results demonstrate the potential for high throughput methods in designing polymer mimics of complex therapeutic proteins, and offer promising leads in the development of better TRAIL-like agents, which are long expected as novel chemotherapies for cancer treatment.

  • (2022) Yu, Tsz Tin
    Thesis
    The rapid emergence and development of antibacterial resistance is a major global threat to public health. There is an urgent need for the development of antibacterial agents with novel therapeutic strategy to tackle the increasing incidence of antibacterial resistance. In recent years, antimicrobial peptides (AMPs) and their synthetic mimics have been under the spotlight of the development of a novel class of antibiotics to combat antibiotic resistance. This research project focused on the utilisation of phenylglyoxamide and benzothiazole scaffolds in the development of antimicrobial peptidomimetics. The synthesis of phenylglyoxamide-based peptidomimetics was achieved via the ring-opening reactions of N-sulfonylisatins with primary amines followed by salt formation. Minimum inhibitory concentrations (MIC) of the peptidomimetics against different bacterial strains were determined to assess their antibacterial activity. Structure-activity relationship (SAR) studies revealed the inverse relationship between the alkylsulfonyl chain length and the bulkiness of the phenyl ring system for high antibacterial activity. The most active peptidomimetics exhibited high antibacterial activity with the lowest MIC to be 4, 16 and 63 μM against S. aureus, E. coli and P. aeruginosa, respectively. These peptidomimetics also showed significant biofilm disruption (up to 50%) and inhibition (up to 70%) against S. aureus at 2–4× MIC. In addition, terphenylglyoxamide-based peptidomimetics synthesised by the ring-opening reaction of N-acylisatins with amines and amino acid esters were evaluated for their quorum sensing inhibition (QSI) activity against P. aeruginosa MH602. The most potent peptidomimetic possessed high QSI activity of 82%, 65% and 53% at 250, 125 and 62.5 μM, respectively, with no bacterial growth inhibition. On the other hand, benzothiazole-based peptidomimetics were synthesised via the Jacobson method of cyclisation of phenylthioamides, followed by the installation of cationic groups. 2-Naphthyl and guanidinium hydrochloride as the hydrophobic and cationic groups, respectively, were important for high antibacterial activity of the peptidomimetics against both Gram-positive and Gram-negative bacteria. The most potent peptidomimetics against S. aureus, E. coli and P. aeruginosa possessed MIC values of 2, 16 and 32 μM, respectively. These active peptidomimetics inhibited 39% of S. aureus biofilm formation and disrupted 42% of preformed S. aureus biofilms at sub-MIC.

  • (2022) Gadde, Satyanarayana
    Thesis
    High-risk neuroblastoma is one of the most aggressive and treatment-refractory childhood malignancies. MYCN (v-myc avian myelocytomatosis viral related oncogene, neuroblastoma derived) is a major oncogenic driver for neuroblastoma (NB) tumorigenesis. Developing direct inhibitors of MYCN has been challenging due to several limitations. Hence, targeting MYCN-binding proteins which regulate the stability of MYCN protein is a promising alternative approach. This study is aimed at developing novel inhibitors of ubiquitin specific protease 5 (USP5), a deubiquitinating enzyme, which is known to prevent MYCN protein degradation by deubiquitination. The first results chapter describes the synthesis of novel pyrido[1,2-a]benzimidazole compounds and their cytotoxicity against MYCN amplified NB cells with high expression of USP5 protein (SK-N-BE(2)-C and Kelly cells). However, none of the tested compounds displayed better cytotoxicity than the parental compound, SE486-11. The second results chapter describes a one-pot synthesis of novel γ-carbolinone, γ-carboline and spiro[pyrrolidinone-3,3′]indoles. One of the γ-carboline compounds (42d) displayed promising cytotoxicity against NB cells (SK-N-BE(2)-C (IC50 = 1.21 μM) and Kelly (IC50 = 3.09 μM)) but showed little therapeutic selectivity when compared to normal human fibroblasts, MRC-5 cells (IC50 = 3.75μM). The synthesis and cytotoxicity of novel pyrimido[1,2-a]benzimidazoles is described in the third results chapter. The active compound, 65a displayed promising cytotoxicity against SK-N-BE(2)-C (IC50 = 0.78 μM) and Kelly (IC50 = 2.00 μM) cells with a reasonable therapeutic window compared to MRC-5 cells (IC50 = 15.0 μM). 65a bound to USP5 protein by microscale thermophoresis assay (Kd = 0.47 µM). USP5 and MYCN protein levels were decreased in NB cells by treatment with 65a. Moreover, the cytotoxicity of 65a was dependant on the expression of USP5 and MYCN proteins. 65a showed synergy in combination with HDAC inhibitors, SAHA and panobinostat. In the fourth results chapter, the synthesis of more potent pyrimido[1,2-a]benzimidazoles with di- and tri- substitutions on the pendant phenyl ring (86b (SK-N-BE(2)-C IC50 = 0.31 μM; Kelly IC50 = 0.65 μM) and 91 (SK-N-BE(2)-C IC50 = 0.03 μM; Kelly IC50 = 0.07 μM)) are described. Importantly, 86b displayed significant in vivo efficacy in TH-MYCN homozygous NB mice when treated with 60 mg/kg for three weeks. The last results chapter describes the synthesis and cytotoxicity of novel benzo[4,5]imidazo[2,1-b]thiazole and pyrido[2,3-b]indole compounds. Collectively, this thesis identifies promising novel scaffolds with great potential for further development.

  • (2022) Chen, Xueqian
    Thesis
    How to design a biosensor with optimal performance with regards to sensitivity, specificity, response time and the ability to be multiplexed is a key issue to meet the demands of diagnostics for health cares. As modern biosensors are usually constructed with bioreceptors immobilized on nanoparticles, the surface of nanoparticle has a significant impact on sensing performance. This thesis aims at investigating how to design the surface of nanoparticle to achieve better sensing performance of single molecule biosensor. Two different biosensing systems were built using DNA functionalized gold nanoparticle (AuNPs), including fluorescent biosensor and localized surface plasmon resonance (LSPR) biosensor using dark-field microscopy. To better understand how the coverage of DNA on surface of AuNPs affects sensing performance, the coverage of DNA on AuNPs surface was adjusted and effect of coverage of DNA on the sensing performance was investigated. Firstly, the impact of coverage of aptamer on the surface of AuNPs on sensing performance was investigated using fluorescence spectroscopy. The number of anti-interferon gamma (IFN-) aptamers was adjusted from an average of 9.6 to 258 per nanoparticle. The sensing strategy was based on the conformational change of anti-IFN- aptamer and fluorescence quenching property of AuNPs. The binding isotherm and binding kinetics of the interaction between AuNPs-aptamer conjugate and IFN- as a function of coverage of aptamer were investigated. It was found that AuNPs-aptamer conjugate with the highest coverage of aptamer was the most favorable in biosensors considering the limit of detection, sensitivity, and response time of detection. Secondly, a LSPR biosensor utilizing AuNPs-DNA conjugates was built to detect SARS-CoV-2 specific RNA. Two AuNPs-DNA conjugates with different size of AuNPs were adopted to form a dimer consisting with a 80 nm gold nanoparticle and a 40 nm gold nanoparticle. The binding of target RNA with dimer resulted in colour change of dimer under dark-field microscope. A calibration curve between response and different concentration of target RNA was obtained with a determined limit of detection of 2.6 fM. Lastly, the number of DNA on the satellite nanoparticle was adjusted by different diluents to optimize the sensing performance of LSPR biosensor. By reducing the amount of DNA on 40 nm gold nanoparticle, the response for the same concentration of target RNA was improved and lower limit of detection was achieved. This thesis provides insights into how to design the surface of nanoparticle and the impact of interfacial design of nanoparticle on the sensing performance of biosensor, which advances the development of single molecule biosensor from the perspective of interface of nanoparticle.

  • (2023) Mustafa, Ahmed
    Thesis
    Polyelectrolytes (PEs) and proteins can spontaneously self-assemble in aqueous solutions to form colloidal polyion complexes. These nanoparticles are widely used in many biotechnological applications such as drug/protein delivery, protein encapsulation and protein stabilisation. Although the interaction between PEs and proteins is mainly electrostatic, it is also influenced by parameters such as the density of negative and positive charges, degree of ionisation, the presence of hydrogen bonding and van der Waals forces exerted by hydrophobic groups throughout the PE and protein, as well as their overall size and concentration. The variety of forces reflects the complexity of the protein's surface where charges are often not evenly distributed, but concentrated in pockets. In order to design polymers that form strong PICs with any given protein, it is necessary to carefully tune the polymer architecture, chain length, side chain chemistry, charge distribution, and hydrophobicity. In this research project, a range of well-defined polymers with different polymeric structures and architectures, copolymerised with a small amount of fluorescent monomer based on the cyanine dye Cy5, were synthesised in low solution volumes without prior deoxygenation using Enz-RAFT polymerisation. The self-assembly of these polymers with Cy3-labelled proteins was screened in order to understand the effect of both polymer and protein structure on binding. Supported by light scattering, isothermal calorimetry (ITC) and small angle X-ray scattering data we demonstrate that a simple plate based Förster resonance energy transfer (FRET) assay can be used as a readout for the binding strength of each polymer to the protein of interest. Because both the synthetic and read-out methodologies are amenable to high throughput processing, this technique enables the rapid combinatorial design of complex polymers for protein encapsulation.

  • (2023) Pelosi, Rosina
    Thesis
    The improvement of luminescent solar concentrator (LSC) efficiency is the core focus of this project. Lead sulfide oleic acid (PbS/OA) semiconductor nanocrystals otherwise known as quantum dots (QDs) are investigated as a potential luminophore in LSCs. Their broad absorption, narrow emission and good Stokes shift are of interest, however cumulative luminophore self-absorption events contribute to losses. A possible way to circumvent these losses is through Förster Resonant Energy Transfer (FRET). In this work PbS/OA QDs of two different sizes are embedded into PMMA films in 1:1 ratios. Mixtures of suitably disparate sized PbS/OA QDs in PMMA films result in photoluminescence (PL) with increased Stokes shifts. Time resolved photoluminescence (TRPL) spectroscopy results are also consistent with FRET, where there is a reduction in high energy QD lifetimes in heterogeneous QD films. PLQY synergies are demonstrated through QD FRET connection in heterogeneous QD spin coated films and improved LSC efficiency is established. This learning may be transferred across other QD species used in LSCs, that also suffer self-absorption losses. The fabrication of a luminescent solar concentrator photovoltaic (LSC/PV) prototype device with dimensions 10 × 10 × 1 cm3 is designed with a commercially available pery- lene luminophore. A power conversion efficiency (PCE) of 22.4 % with a 2.4% increase on PV alone is demonstrated and attributable to the LSC through novel experimental measurement design. Furthermore, it was shown that a parabolic patterned textured luminescent film had a threefold increase in emission over a flat film. The parabolic design within the film was able to take broadband light on one side and augment emission preferentially out the opposite side. The film performance permits up to a threefold cost benefit in real world applications, making costly green-housing films that emit in the photosynthetically active region more accessible. Finally, future directions and pathways for green technology LSCs are discussed within a New Product Development (NPD) framework.

  • (2023) Steller, Luke
    Thesis
    Boron can form an unique stabilizing complex with ribose, the backbone of RNA, and is therefore considered to be an essential element in the emergence of life. However, the availability of boron in actualistic settings proposed for the emergence of life are poorly understood. This thesis investigates the processes that may have contributed to the concentration and aqueous chemical availability of boron in early Earth environments and, in particular, the potential role these processes may have played in an emergence of life scenario in terrestrial hot springs. This investigation was achieved through a combination of geological, geochemical, and experimental approaches that explored deep time Earth settings, modern analogues and prebiotic chemical systems. First, this thesis explored the concentration and isotopic fractionation of boron in Archean seawater, as Archean seawater boron concentration is poorly constrained and available estimates for B isotopic fractionation include very large errors that preclude the ability to provide geochemical constraints for an important end–member fluid for emergence of life scenarios. In Chapter 2, the concentration and isotopic fractionation of early Archean seawater was estimated using a new proxy for Archean seawater in the form of bladed calcite discovered exclusively within the interpillow spaces of two separate submarine basalt units from the Pilbara Supergroup, the 3.49 Ga North Star Basalt and 3.35 Ga Euro Basalt. Bladed calcite is a texture well known from the economic geology literature as having precipitated from boiling seawater trapped in the interpillow spaces of the pillow basalts during their eruption. Geochemical analysis show that bladed calcite samples had varied boron concentrations (0.2 to 2 ppm), and δ11B values (–6‰ to 8.5‰). After samples that were impacted by contamination or magmatic fluids were removed from the study, previously published experimental data that accounted for the fractionation induced by calcite precipitate at elevated temperatures was applied to determine a proxy for parent fluid chemistry. This resulted in an estimate for Archean seawater δ11B value of 15.9 ± 3.8‰ and 18.3 ± 5.2‰, at 3.49 Ga and 3.35 Ga, respectively, with an average aqueous boron concentration of 1.5 ± 1.3 ppm. These values not only fill a 500–million–year gap in Archean seawater boron estimates but, when combined with previous estimates, also point to a secular trend of increasing δ11B values throughout the Archean. The results provided in Chapter 2 indicate that seawater boron concentrations in the oceans was likely insufficient to stabilize ribose formation on early Earth, and that additional processes were required to concentrate boron at levels sufficient to promote prebiotic chemistry leading to the emergence of life. To develop a better understanding of the geological processes that could concentrate boron in early Earth environments, Chapter 3 reviews and collates previous studies on the concentration and isotopic fractionation of boron on the early Earth, and specifically within the hot spring system preserved within the 3.5 Ga Dresser Formation of the Pilbara Craton, the host to the oldest, most convincing evidence of life. This summary, when combined with the findings of Chapter 2, is used to develop a whole–system model of boron isotope fractionation for a realistic early Earth emergence of life setting in a volcanic plateau, where magmatic fluid fractionation, combined with absorption onto minerals and surficial evaporation, was able to drive boron concentration to suitable values for prebiotic ribose stabilization on explosed land surface. However, to apply the boron concentration findings proposed in Chapter 3 to a model of the prebiotic Earth, certain factors that were present on the Earth before life formed, including the abundance of abiological organic molecules, should be considered. To investigate this, Chapter 4 conducted experiments where a range of borate–bearing minerals (including authentic hot spring deposits) were exposed to ribose to determine its impact on borate mineral solubility and chemical availability. Results show that borate mineral solubility was significantly increased by ribose in alkaline conditions, even in the presence of calcium, a cation that normally removes borate from solution as an insoluble precipitate. These experiments confirm that variable pH environments (fluctuating between alkaline and acidic conditions) can both promote ribose–borate complexation, along with subsequent ribose release, allowing ribose to participate in further prebiotic chemical reactions. The results demonstrate that prebiotic organic molecules have the ability to significantly alter the solubility of borate minerals, thereby increasing our understanding of the potential availability of aqueous boron on prebiotic Earth. Building on the previous chapter’s findings that specific environmental conditions in surficial settings (i.e., evaporation and variable pH) are required to promote boron accumulation and availability on prebiotic Earth, experiments conducted as part of Chapter 5 investigated the impact of evaporation and variable pH environments on the formation of lipid vesicles capable of organic macromolecule (including RNA) encapsulation. Results demonstrate that a single wet/dry cycle driven by evaporation can remodel dense lipid aggregates into thin–walled vesicles capable of macromolecule encapsulation, even under acidic pH conditions, expanding the previously known pH limits for vesicle formation. Furthermore, it was found that wet/dry cycles appear to favor the encapsulation of RNA within thin–walled vesicles relative to more dense vesicles, thus conferring thin–walled protocells a selective advantage. The findings presented in this thesis provide a deeper understanding for the geochemical processes that may have promoted boron concentration and availability in an origins of life scenario, and demonstrate the importance of considering the impact that these environmental processes may have on other proposed prebiotic mechanisms (i.e., protocell formation). These results help support the hypothesis that surficial hot spring systems were a likely setting for the emergence of life on Earth.