New phosphorescence based probes and techniques for the analysis of cellular oxygen and respiration

Real time monitoring of oxygenation and respiration is on the cutting edge of bioanalysis, including studies of cell metabolism, bioenergetics, mitochondrial function and drug toxicity. This thesis presents the development and evaluation of new luminescent probes and techniques for intracellular O2 sensing and imaging. A new oxygen consumption rate (OCR) platform based on the commercial microfluidic perfusion channel μ-slides compatible with extra- and intracellular O2 sensitive probes, different cell lines and measurement conditions was developed. The design of semi-closed channels allowed cell treatments, multiplexing with other assays and two-fold higher sensitivity to compare with microtiter plate. We compared three common OCR platforms: hermetically sealed quartz cuvettes for absolute OCRs, partially sealed with mineral oil 96-WPs for relative OCRs, and open 96-WPs for local cell oxygenation. Both 96-WP platforms were calibrated against absolute OCR platform with MEF cell line, phosphorescent O2 probe MitoXpress-Intra and time-resolved fluorescence reader. Found correlations allow tracing of cell respiration over time in a high throughput format with the possibility of cell stimulation and of changing measurement conditions. A new multimodal intracellular O2 probe, based on the phosphorescent reporter dye PtTFPP, fluorescent FRET donor and two-photon antennae PFO and cationic nanoparticles RL-100 was described. This probe, called MM2, possesses high brightness, photo- and chemical stability, low toxicity, efficient cell staining and high-resolution intracellular O2 imaging with 2D and 3D cell cultures in intensity, ratiometric and lifetime-based modalities with luminescence readers and FLIM microscopes. Extended range of O2 sensitive probes was designed and studied in order to optimize their spectral characteristics and intracellular targeting, using different NPs materials, delivery vectors, ratiometric pairs and IR dyes. The presented improvements provide useful tool for high sensitive monitoring and imaging of intracellular O2 in different measurement formats with wide range of physiological applications.

Toxicity of manufactured nano-ZnO to Lemna minor

The rapid development of nanotechnology has led to a rise in the large-scale production and commercial use of engineered nano-ZnO. Engineered/manufactured nano-ZnO are applied in a broad range of products such as drugs, paints, cosmetics, abrasive agents and insulators. This can result in the unintended exposure of human beings to nano-ZnO and will inevitably result in the release of nano-ZnO in to the environment. Thus, it is necessary to assess the risk of nano-ZnO to the environment. In this thesis the toxicity of nano-ZnO was analysed using the aquatic, primary producer lesser duckweed (Lemna minor), and the mechanism of toxicity was analysed. Both short-term (one week) and long-term (six weeks) toxicity of nano-ZnO (uncoated) were determined. Results show that the toxicity of nano-ZnO added to the aquatic growth medium increases with increasing concentration and that toxicity accumulates with exposure time. A study of nano-ZnO dissolution reveals that the main reason for nano-ZnO toxicity on Lemna minor is the release of Zn ions. Nano-ZnO dissolution is pH dependent, and toxicity matches the release of Zn2+. Functional coating materials are commonly added to nano-ZnO particles to improve specific industrial applications. To test if coating materials contribute to nano-ZnO toxicity on lesser duckweed, the effect of silane coupling agent (KH550) coated nano-ZnO on Lemma minor was investigated. Results show that coating can decrease the release of Zn ions, which reduces toxicity to Lemna minor, in contrast to uncoated particles. Another commonly hypothesized reason for nano-ZnO toxicity is the formation of Reactive Oxygen Species (ROS) on the particles surface. As part of this thesis, the ROS formation induced by nano-ZnO was studied. Results show that nano-ZnO catalyse ROS formation and this can negatively affect duckweed growth. In conclusion, this work has detailed potentially toxic effects of nano-ZnO on Lemna minor. This study has also provides references for future research, and informs regulatory testing for nanoparticle toxicity. Specifically, the outcomes of this study emphasize the importance of exposure time, environmental parameters and coating material when analysing NPs toxicity. Firstly, impacts of longer exposure time should be studied. Secondly, environmental parameters such as pH and medium-composition need to be considered when investigating NPs toxicity. Lastly, coating of NPs should always be considered in the context of NPs toxicity, and similar NPs with different coatings require separate toxicity tests.

A study of the oxygen electrochemistry of ruthenium and iridium

This thesis is concerned with an investigation of the anodic behaviour of ruthenium and iridium in aqueous solution and particularly of oxygen evolution on these metals. The latter process is of major interest in the large-scale production of hydrogen gas by the electrolysis of water. The presence of low levels of ruthenium trichloride ca. 10-4 mol dm-3 in acid solution give a considerable increase in the rate of oxygen evolution from platinum and gold, but not graphite, anodes. The mechanism of this catalytic effect was investigated using potential step and a.c. impedance technique. Earlier suggestions that the effect is due to catalysis by metal ions in solution were proved to be incorrect and it was shown that ruthenium species were incorporated into the surface oxide film. Changes in the oxidation state of these ruthenium species is probably responsible for the lowering of the oxygen overvoltage. Both the theoretical and practical aspects of the reaction were complicated by the fact that at constant potential the rates of both the catalysed and the uncatalysed oxygen evolution processes exhibit an appreciable, continuous decrease with either time or degree of oxidation of the substrate. The anodic behaviour of iridium in the oxide layer region has been investigated using conventional electrochemical techniques such as cyclic voltammetry. Applying a triangular voltage sweep at 10 Hz, 0.01 to 1.50V increases the amount of electric charge which the surface can store in the oxide region. This activation effect and the mechanism of charge storage is discussed in terms of both an expanded lattice theory for oxide growth on noble metals and a more recent theory of irreversible oxide formation with subsequent stoichiometry changes. The lack of hysteresis between the anodic and cathodic peaks at ca. 0.9 V suggests that the process involved here is proton migration in a relatively thick surface layer, i.e. that the reaction involved is some type of oxide-hydroxide transition. Lack of chloride ion inhibition in the anodic region also supports the irreversible oxide formation theory; however, to account for the hydrogen region of the potential sweep a compromise theory involving partial reduction of the outer regions of iridium oxide film is proposed. The loss of charge storage capacity when the activated iridium surface is anodized for a short time above ca. 1.60 V is attributed to loss by corrosion of the outer active layer from the metal surface. The behaviour of iridium at higher anodic potentials in acid solution was investigated. Current-time curves at constant potential and Tafel plots suggested that a change in the mechanism of the oxygen evolution reaction occurs at ca. 1.8 V. Above this potential, corrosion of the metal occurred, giving rise to an absorbance in the visible spectrum of the electrolyte (λ max = 455 nm). It is suggested that the species involved was Ir(O2)2+. A similar investigation in the case of alkaline electrolyte gave no evidence for a change in mechanism at 1.8 V and corrosion of the iridium was not observed. Oxygen evolution overpotentials were much lower for iridium than for platinum in both acidic and alkaline solutions.

Use of oxygen sensors for the non destructive measurement of oxygen in packaged food and beverage products and its impact on product quality and shelf life

The principal objective of this thesis was to investigate the ability of reversible optical O2 sensors to be incorporated into food/beverage packaging systems to continuously monitor O2 levels in a non-destructive manner immediately postpackaging and over time. Residual levels of O2 present in packs can negatively affect product quality and subsequently, product shelf-life, especially for O2-sensitive foods/beverages. Therefore, the ability of O2 sensors to continuously monitor O2 levels present within food/beverage packages was considered commercially relevant in terms of identifying the consequences of residual O2 on product safety and quality over time. Research commenced with the development of a novel range of O2 sensors based on phosphorescent platinum and palladium octaethylporphyrin-ketones (OEPk) in nano-porous high density polyethylene (HDPE), polypropylene (PP) polytetrafluoroethylene (PTFE) polymer supports. Sensors were calibrated over a temperature range of -10°C to +40°C and deemed suitable for food and beverage packaging applications. This sensor technology was used and demonstrated itself effective in determining failures in packaging containment. This was clearly demonstrated in the packaging of cheese string products. The sensor technology was also assessed across a wide range of packaged products; beer, ready-to-eat salad products, bread and convenience-style, muscle-based processed food products. The O2 sensor technology performed extremely well within all packaging systems. The sensor technology adequately detected O2 levels in; beer bottles prior to and following pasteurisation, modified atmosphere (MA) packs of ready-to-eat salad packs as respiration progressed during product storage and MA packs of bread and convenience-style muscle-based products as mycological growth occurred in food packs over time in the presence and absence of ethanol emitters. The use of the technology, in conjunction with standard food quality assessment techniques, showed remarkable usefulness in determining the impact of actual levels of O2 on specific quality attributes. The O2 sensing probe was modified, miniaturised and automated to screen for the determination of total aerobic viable counts (TVC) in several fish species samples. The test showed good correlation with conventional TVC test (ISO:4833:2003), analytical performance and ruggedness with respect to variation of key assay parameters (probe concentration and pipetting volume). Overall, the respirometric fish TVC test was simple to use, possessed a dynamic microbial range (104-107 cfu/g sample), had an accuracy of +/- one log(cfu/g sample) and was rapid. Its ability to assess highly perishable products such as fish for total microbial growth in <12 hr demonstrates commercial potential.

HIF-1alpha role in oxygen-dependent radio- and chemosensitivity

Poor oxygenation (hypoxia) is a common characteristic of human solid tumours, and is associated with cell survival, metastasis and resistance to radio- and chemotherapies. Hypoxia-induced stabilisation of hypoxia-inducible factor-1α (HIF-1α) leads to changes in expression of various genes associated with growth, vascularisation and metabolism. However whether HIF-1α plays a causal role in promoting hypoxic resistance to antitumour therapies remains unclear. In this study we used pharmacological and genetic methods to investigate the HIF-1α contribution to radio- and chemoresistance in four cancer cell lines derived from cervical, breast, prostate and melanoma human tumours. Under normoxia or hypoxia (<0.2% or 0.5% oxygen) the cells were exposed to either a standard irradiation dose (6.2 Gy) or chemotherapeutic drug (cisplatin), and subsequent cell proliferation (after 7 days) was measured in terms of resazurin reduction. Oxygen-dependent radio- and chemosensitivity was evident in all wild type whereas it was reduced or abolished in HIF-1α (siRNA) knockdown cells. The effects of HIF-1α-modulating drugs (EDHB, CoCl2, deferoxamine to stabilise and R59949 to destabilise it) reflected both HIF-1α-dependent and independent mechanisms. Collectively the data show that HIF-1α played a causal role in our in vitro model of hypoxia-induced radioresistance whereas its contribution to oxygendependent sensitivity to cisplatin was less clear-cut. Although this behavior is likely to be conditioned by further biological and physical factors operating in vivo, it is consistent with the hypothesis that interventions directed at HIF-1α may improve the clinical effectiveness of tumour treatments.