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(2022) Li, YiThesisCRISPR/Cas9-based gene editing is no doubt among the most intensively studied topics in bio-related fields in the recent decade, and certain new programmable Cas nucleases have been exploited recently for the development of a variety of biological tools far beyond gene editing, particularly for biosensor development. Although CRISPR/Cas-based biosensing has brought about a revolution in the area of nucleic acid diagnostic with their superior performances, its advantages were challenged when attempting to expand towards a broader range of non-nucleic acid targets. The currently reported methods for non-nucleic acid targets has successfully demonstrated the versality of CRISPR/Cas components in combining with other biosensing elements, however, combining these elements without proper optimization or controllable bioreaction environments could also bring additional variable factors into the system, hence potentially leading to compromised sensitivity or overall increase of system complexity. In this project, two novel CRISPR/Cas12a-based biosensing systems have been developed to realise simple, sensitive and universal non-nucleic acid detections. Both of these systems are established on a standard 96-well plate format, similar to the widely used ELISA diagnostic approach. The first system utilised an aptamer as its recognition molecule for rapid detection of two small proteins with fM-level sensitivity within 1.5 hours. In the second system, antibody was used as a recognition molecule, allowing to draw on a huge pool of commercially available antibodies to support its universality. This system exhibits ultra-high sensitivity down to 1 fg/mL (aM-level) for the detection of two protein targets. With these two successfully developed CRISPR/Cas12a-based non-nucleic acid biosensing systems, the universality and feasibility to deal with two practical bio-detection scenarios was investigated. The antibody-based system with minor modifications was directly used as a ready-to-use sensitivity enhancer onto a commercial IFN-γ ELISA kit, which resulted in a 2-log increase in sensitivity without changing its original protocol. Then, a similar modification strategy was used to re-direct the antibody-based system to detect whole pathogenic microorganism, Cryptosporidium parvum oocyst. Without the need for specialised instruments, the results show a successful detection of this pathogen with single oocyst sensitivity and capable of applying in challenging environmental samples. All these results serve as successful demonstration of the great potential in CRISPR/Cas-based biosensing technology to achieve affordable, translatable, and deployable solutions for various clinical, industrial and research diagnostic needs.
(2022) Cao, JunThesisThis thesis focuses on the development and applications of magnetic resonance electrical properties tomography (MREPT), which is an emerging imaging modality to noninvasively obtain the electrical properties of tissues, such as conductivity and permittivity. Chapter 2 describes the general information about human research ethics, MRI scanner, MR sequence and the method of phase-based MREPT implemented in this thesis. Chapter 3 examines the repeatability of phase-based MREPT in the brain conductivity measurement using balanced fast field echo (bFFE) and turbo spin echo (TSE) sequences, and investigate the effects of compressed SENSE, whole-head B_1 shimming and video watching during scan on the measurement precision. Chapter 4 investigates the conductivity signal in response to short-duration visual stimulus, compares the signal and functional activation pathway with that of BOLD, and tests the consistency of functional conductivity imaging (funCI) with visual stimulation across participants. Chapter 5 extends the use of functional conductivity imaging to somatosensory stimulation and trigeminal nerve stimulation to evaluate the consistency of functional conductivity activation across different types of stimuli. In addition, visual adaptation experiment is performed to test if the repetition suppression effect can be observed using funCI. Chapter 6 explores if resting state conductivity networks can be reliably constructed using resting state funCI, evaluates the consistency of persistent homology architectures, and compares the links between nodes in the whole brain. Chapter 7 investigates the feasibility of prostate conductivity imaging using MREPT, and distinctive features in the conductivity distribution between healthy participants and participants with suspected abnormalities.
(2022) Kaur, JagjitThesisSecreted by pancreatic β-cells, insulin is the major anabolic hormone, regulating the metabolism of fats, proteins, and carbohydrates. Defects in insulin production or action can lead to diabetes characterized by derangements in glucose handling and metabolic disease. Diabetes affects 420 million people worldwide, increasing morbidity, mortality and placing a burden on healthcare of nations. There is a need for rapid and accurate monitoring of insulin levels to optimize diabetes management and facilitate early diagnosis of insulin related chronic diseases. Conventional strategies such as HPLC, MALDI-TOF, ELISA, etc. used for insulin detection are not suitable for point-of-care testing (POCT) as they are expensive, and require sample preparation, sophisticated instruments, and skilled personnel. Our goal was to develop techniques to allow POCT for insulin in real time. In this study we developed two lateral flow assays (LFAs) based POCT platforms using aptamers as the biorecognition molecules for colorimetric and fluorescent detection of insulin. A range of conditions were tested such as concentrations of aptamers, reporter molecules used, volume of sample required, and assay time to obtain quantify insulin levels using a standard LFA reader. The colorimetric LFAs had linear detection range of 0.01-1 ng.mL-1 and LOD of 0.01 ng.mL-1. The fluorescent LFAs exhibited a linear detection range of 0-4 ng.mL-1 and 0.1 ng.mL-1 LOD. Various signal amplification strategies were incorporated, ie., gold-silver amplification technique and rolling circular amplification (RCA) to further enhance the signal. The developed colorimetric LFAs were successfully used for insulin quantification in rat blood, human blood, and human saliva samples. Although insulin levels were quantified within 12 min, some issues arose such as coagulation, need for dilution, and non-uniform flow through the test strips. Further work is required to optimize blood handling to progress an insulin POCT in real time. Future work could develop a multiplexed strip for detection of different analytes such as HbA1c, glucose, and C-peptide for better management of diabetes, along with a smartphone reader App. This research goes some way to addressing the challenge of providing a reliable and rapid approach for highly sensitive and specific detection of insulin for POCT applications.
(2006) Doran, MichaelThesisThe transplantation of ex vivo expanded mobilized peripheral blood haematopoietic stem cells (PBSC), in place of unmanipulated cells following high dose chemotherapy, reduces the period of cytopenia associated with the therapy’s hemotoxicity. In this thesis the development and optimization of a preclinical prototype hollow fiber bioreactor (HFBR) for the ex vivo expansion of PBSC is described. Mass transport measurements and model of metabolite profiles demonstrate that Cuprophan and Polyflux are suitable membrane material for high-density cell expansion in a HFBR. Materials selected for the HFBR were found to be non-toxic following a 20-day saline extraction. Growth factor (GF) adsorption to the Polyflux membrane makes it unsuitable for expansion of GF dependent cells. However, the GF retention and minimal adsorption characteristics of the Cuprophan membrane are appropriate for this application. Cell-free medium degrades at 37ºC by an oxygen dependent process generating byproducts that inhibit cell growth. This process is relevant to perfusion bioreactors where the bulk of the medium is maintained at 37ºC and is cell-free. Albumin was shown to slow the degradation process but was itself degraded by shear damage inflicted during recirculation. Treating recirculating medium with dialysis against albumin was shown to be a more effective way to mitigate the effects of degradation and lengthen the functional life of albumin over conventional suspension of albumin in the recirculating medium. The preclinical prototype HFBR utilised dialysis against albumin to expand KG-1a cultures from densities as low as 3.5x10^5 cells/ml up to as high as 2x10^8 cells/ml with expansion rates equivalent to T-flask cultures. This process was then applied to PBSC where the targeted 100-fold expansion was achieved. Process optimization was continued using cord blood (CB) CD34+ cells. Growth factor loading sufficient for PBSC expansion in the HFBR was inadequate for CB expansions due to greater than anticipated CB uptake rates. The cell product from the HFBR contained significantly greater yields of CD34+ cells than attained using T-flask cultures. The HFBR platform is suitable for PBSC expansion and appears promising for CB expansion.
(2022) Qiao, LaicongThesisThere 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
Nanomaterials and Polymers for Nitric Oxide Therapeutic Delivery and Sensing of Nitric Oxide Reservoir(2022) Luo, JeffThesisNitric oxide (NO) plays pivotal roles in various physiological systems and has immense therapeutic potential. NO, however, has a short half-life (<5 s) and a short diffusion distance of ~160 μm in vivo, and its physiological functions are highly dependent on its concentrations. Current NO delivery strategies can be generally categorized into non-catalytic and catalytic (enzymatic) approaches. For the former, the longevity of the NO delivery systems principally relies on the finite NO donor reservoir, while the latter is limited by the low stability of natural enzymes. Another important challenge in NO delivery is the difficulty in accurately detecting circulating NO reservoir in blood. To address these challenges, this thesis focuses on the design, synthesis, and applications of nano-biomaterials to enable sustained NO delivery and accurate detection of endogenous circulating NO reservoir. This thesis revealed ceria nanoparticles as a new class of nanomaterials with the unique ability to catalyze NO generation from NO donors. The therapeutic activity of ceria-induced NO was demonstrated to inhibit cancer cell proliferation. This unique NO-generating feature stood in contrast to the well-established understanding of ceria to scavenge NO. This study provided deeper insights into the bio-functions of ceria nanoparticles and broadened their biomedical applications. Then this thesis reported the first catalytic polymers that generate NO, in particular amine-containing polymers, e.g., polyethyleneimine (PEI). These polymers can be easily integrated into a suite of biomaterials (e.g. hydrogels) to equip them with NO delivery capability. The therapeutic application of polymer-induced NO was demonstrated to prevent the formation of Pseudomonas aeruginosa biofilm. Finally, the thesis tackled the demand for rapid and accurate detection of human serum albumin (HSA, the most abundant circulating NO reservoir in blood) by developing a fluorescent paper-based sensor. This sensing platform allowed sensitive (detection limit of 0.91 g/L) and rapid (20 minutes) point-of-care detection of HSA and HSA-related disease diagnosis by visible color change, and could be extended to the detection of a spectrum of biomarkers. Collectively, these findings open new routes to produce next generation nano-biomaterials for the diverse biomedical applications of NO such as anticancer, antibacterial, and sensing applications.