Immunoassays for the detection of illicit drugs and organophosphate pesticides

The research presented in this thesis describes the production and application of antibodies against morphine-3-glucuromde and parathion, an organophosphate pesticide. A variety of morphine-3-glucuronide-protein conjugates were produced and used to generate and characterise polyclonal antibodies. These polyclonal antibodies were used in the development of immunoassays in a competitive ELISA format and on the BIAcore (a surface plasmon resonance based optical biosensor capable of monitoring biomolecular interactions in ‘real-time’). Combinatorial single chain Fv (scFv) antibody phage display libraries were also generated to morphine-3-glucuromde Splenomic mRNA from mice pre-immunised with a morphine-3-glucuronide-BSA conjugate was used in the amplification of antibody genes followed by cloning into vectors from a well-established phage display system. Two positive clones were isolated in the first two rounds of panning. One clone (E3) was selected and re-cloned into a plasmid for soluble scFv antibody expression. The soluble scFv antibody was purified and used in the development of a competitive ELISA-based assay. Further analysis of the E3 clone was carried out in the development of an inhibition BIAcore assay for morphine-3-glucuromde in biological matrices Affinity determinations of the scFv antibody to morphine-3- glucuromde were carried out using ‘real-time’ biomolecular interaction analysis. Polyclonal antibodies to the organophosphate pesticide, parathion, were also produced and characterised. They were used to generate competitive ELISA and BIAcore-based inhibition immunoassays However, when analysed on the BIAcore, the parathion polyclonal antibody displayed no inhibition

Applications for Gradient Metal Alloys Fabricated Using Additive Manufacturing

Recently, additive manufacturing (AM) techniques have been developed that may shift the paradigm of traditional metal production by allowing complex net-shaped hardware to be built up layer-by-layer, rather than being machined from a billet. The AM process is ubiquitous with polymers due to their low melting temperatures, fast curing, and controllable viscosity, and 3D printers are widely available as commercial or consumer products. 3D printing with metals is inherently more complicated than with polymers due to their higher melting temperatures and reactivity with air, particularly when heated or molten. The process generally requires a high-power laser or other focused heat source, like an electron beam, for precise melting and deposition. Several promising metal AM techniques have been developed, including laser deposition (also called laser engineered net shaping or LENS and laser deposition technology (LDT)), direct metal laser sintering (DMLS), and electron beam free-form (EBF). These machines typically use powders or wire feedstock that are melted and deposited using a laser or electron beam. Complex net-shape parts have been widely demonstrated using these (and other) AM techniques and the process appears to be a promising alternative to machining in some cases. Rather than simply competing with traditional machining for cost and time savings, the true advantage of AM involves the fabrication of hardware that cannot be produced using other techniques. This could include parts with "blind" features (like foams or trusses), parts that are difficult to machine conventionally, or parts made from materials that do not exist in bulk forms. In this work, the inventors identify that several AM techniques can be used to develop metal parts that change composition from one location in the part to another, allowing for complete control over the mechanical or physical properties. This changes the paradigm for conventional metal fabrication, which relies on an assortment of "post-processing" methods to locally alter properties (such as coating, heat treating, work hardening, shot peening, etching, anodizing, among others). Building the final part in an additive process allows for the development of an entirely new class of metals, so-called "functionally graded metals" or "gradient alloys." By carefully blending feedstock materials with different properties in an AM process, hardware can be developed with properties that cannot be obtained using other techniques but with the added benefit of the net-shaped fabrication that AM allows

Breakdown of the semiclassical approximation during the early stages of preheating

The validity of the semiclassical approximation is investigated during the preheating phase in models of chaotic inflation using a modification of a criterion previously proposed for semiclassical gravity. If the modified criterion is violated then fluctuations of the two-point function for the quantum elds are large and the semiclassical approximation is not valid. Evidence is provided that the semiclassical approximation breaks down during the early stages of preheating, well before either scattering effects or backreaction effects are important

Fv antibodies to aflatoxin B1 derived from a pre-immunized antibody phage display library system

The production and characterization of recombinant antibodies to aflatoxin B[SUB1] (AFB[SUB1]), a potent mycotoxin and carcinogen is described. The antibody fragments produced were then applied for use in a surface plasmon resonance-based biosensor (BIAcore), which measures biomolecular interactions in 'real-time'. Single chain Fv (scFv) antibodies were generated to aflatoxin B1 from an established phage display system, which incorporated a range of different plasmids for efficient scFv expression. The scFv's were used in the development of a competitive ELISA, and also for the development of surface plasmon resonance (SPR)-based inhibition immunoassays. They were found to be suitable for the detection of AFB[SUB1], in this format, with the assays being sensitive and reproducible

Granular Media-Based Tunable Passive Vibration Suppressor

and vibration suppression device is composed of statically compressed chains of spherical particles. The device superimposes a combination of dissipative damping and dispersive effects. The dissipative damping resulting from the elastic wave attenuation properties of the bulk material selected for the granular media is independent of particle geometry and periodicity, and can be accordingly designed based on the dissipative (or viscoelastic) properties of the material. For instance, a viscoelastic polymer might be selected where broadband damping is desired. In contrast, the dispersive effects result from the periodic arrangement and geometry of particles composing a linear granular chain. A uniform (monatomic) chain of statically compressed spherical particles will have a low-pass filter effect, with a cutoff frequency tunable as a function of particle mass, elastic modulus, Poisson fs ratio, radius, and static compression. Elastic waves with frequency content above this cutoff frequency will exhibit an exponential decay in amplitude as a function of propagation distance. System design targeting a specific application is conducted using a combination of theoretical, computational, and experimental techniques to appropriately select the particle radii, material (and thus elastic modulus and Poisson fs ratio), and static compression to satisfy estimated requirements derived for shock and/or vibration protection needs under particular operational conditions. The selection of a chain of polymer spheres with an elastic modulus .3 provided the appropriate dispersive filtering effect for that exercise; however, different operational scenarios may require the use of other polymers, metals, ceramics, or a combination thereof, configured as an array of spherical particles. The device is a linear array of spherical particles compressed in a container with a mechanism for attachment to the shock and/or vibration source, and a mechanism for attachment to the article requiring isolation (Figure 1). This configuration is referred to as a single-axis vibration suppressor. This invention also includes further designs for the integration of the single-axis vibration suppressor into a six-degree-of-freedom hexapod "Stewart"mounting configuration (Figure 2). By integrating each singleaxis vibration suppressor into a hexapod formation, a payload will be protected in all six degrees of freedom from shock and/or vibration. Additionally, to further enable the application of this device to multiple operational scenarios, particularly in the case of high loads, the vibration suppressor devices can be used in parallel in any array configuration

Development of a decision support tool to facilitate primary care management of patients with abnormal liver function tests without clinically apparent liver disease [HTA03/38/02]. Abnormal Liver Function Investigations Evaluation (ALFIE)

Liver function tests (LFTs) are routinely performed in primary care, and are often the gateway to further invasive and/or expensive investigations. Little is known of the consequences in people with an initial abnormal liver function (ALF) test in primary care and with no obvious liver disease. Further investigations may be dangerous for the patient and expensive for Health Services. The aims of this study are to determine the natural history of abnormalities in LFTs before overt liver disease presents in the population and identify those who require minimal further investigations with the potential for reduction in NHS costs

Dissolved Organic Carbon Cycling in Forested Watersheds: A Carbon Isotope Approach

Dissolved organic carbon (DOC) is important in the acid‐base chemistry of acid‐sensitive freshwater systems; in the complexation, mobility, persistence, and toxicity of metals and other pollutants; and in lake carbon metabolism. Carbon isotopes (13C and 14C) are used to study the origin, transport, and fate of DOC in a softwater catchment in central Ontario. Precipitation, soil percolates, groundwaters, stream, beaver pond, and lake waters, and lake sediment pore water were characterized chemically and isotopically. In addition to total DOC, isotopic measurements were made on the humic and fulvic DOC fractions. The lake is a net sink for DOC. Δ14C results indicate that the turnover time of most of the DOC in streams, lakes, and wetlands is fast, less than 40 years, and on the same time scale as changes in acidic deposition. DOC in groundwaters is composed of older carbon than surface waters, indicating extensive cycling of DOC in the upper soil zone or aquifer

Mechanical Properties of Molybdenum Disulfide and the Effect of Doping: An in Situ TEM Study

Direct observations on nanopillars composed of molybdenum disulfide (MoS₂) and chromium-doped MoS₂ and their response to compressive stress have been made. Time-resolved transmission electron microscopy (TEM) during compression of the submicrometer diameter pillars of MoS₂- and Cr-doped MoS₂ (Cr: 0, 10, and 50 at %) allow the deformation process of the material to be observed and can be directly correlated with mechanical response to applied load. The addition of chromium to the MoS₂ changed the failure mode from plastic deformation to catastrophic brittle fracture, an effect that was more pronounced as chromium content increased

Castable Bulk Metallic Glass Strain Wave Gears: Towards Decreasing the Cost of High-Performance Robotics

The use of bulk metallic glasses (BMGs) as the flexspline in strain wave gears (SWGs), also known as harmonic drives, is presented. SWGs are unique, ultra-precision gearboxes that function through the elastic flexing of a thin-walled cup, called a flexspline. The current research demonstrates that BMGs can be cast at extremely low cost relative to machining and can be implemented into SWGs as an alternative to steel. This approach may significantly reduce the cost of SWGs, enabling lower-cost robotics. The attractive properties of BMGs, such as hardness, elastic limit and yield strength, may also be suitable for extreme environment applications in spacecraft