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Biological O2 sensing probes and measurement techniques were first introduced in the late 80s. In the last 3-5 years they have undergone major development that have made them available and affordable for a broad range of applications in various disciplines of the life and biomedical sciences. These new chemistries and technologies, which are significantly different from the majority of other fluorescence-based probes and detection techniques, have already demonstrated their high utility. This book will provide a systematic overview of the existing and emerging O2 sensing technologies in their different modifications, a practical guide to their rational selection and use, and examples of biological applications/case studies, including details on how to set up and conduct such experiments, troubleshoot and interpret the data.
Significant progress has been made in recent years in quenched-phosphorescence oxygen sensing, particularly in the materials and applications of this detection technology that are open to commercialization, like uses in brain imaging and food packaging. Prompted by this, the editors have delivered a dedicated book that brings together these developments, provides a comprehensive overview of the different detection methodologies, and representative examples and applications. This book is intended to attract new researchers from various disciplines such as chemistry, physics, biology and medicine, stimulate further progress in the field and assist in developing new applications. Providing a concise summary at the cutting edge, this practical guide for current experts and new potential users will increase awareness of this versatile sensing technology.
Time-correlated Single Photon Counting has been written in the hope that by relating the authors' experiences with a variety of different single photon counting systems, they may provide a useful service to users and potential users of this formidably sensitive technique. Of all the techniques available to obtain information on the rates of depopulation of excited electronic singlet states of molecular species, monitoring of fluorescence provides, in principle, the simplest and most direct measure of concentration. This volume comprises eight chapters, with the first focusing on the time dependence and applications of fluorescence. Succeeding chapters go on to discuss basic principles of the single photon counting lifetime measurement; light sources; photomultipliers; electronics; data analysis; nanosecond time-resolved emission spectroscopy; time dependence of fluorescence anisotropy. This book will be of interest to practitioners in the field of chemistry.
Analysis of biological oxygen uptake can give information on the general metabolic state of test cells, specific information on mitochondrial function, and allow direct analysis of the activity of oxygen dependant enzymes. Presented in this thesis is the development for such analysis. A range of existing and new phosphorescent Pt(II)-porphyrins based oxygen sensitive probes are studied. Water soluble macromolecular probes were produced through the covalent labelling of a carrier (polypeptide, polysaccharide or PEG) with a monofunctional isothiocyanate derivative of Pt(II) coproporphyrin. Parameters effecting water-soluble probe performance were evaluated, including the effect of the macromolecular carrier and labelling ratio. Oxygen sensitive microparticle probes were also produced. Both probe types can be added to test sample and analysed on standard plate readers. A number of different measurement formats are evaluated, including standard micro-well plates and glass capillaries. The development and evaluation of a dedicated low volume platform for respirometric measurements is also presented. Using these probes and measurement formats, the oxygen uptake of various suspension and adherent cell types, and the activity of oxygen dependent enzymes were successfully measured. This measurement approach was applied to the analysis of cytotoxicity. Cells were treated with known toxins and the effect on oxygen uptake monitored. Oxygen uptake was seen to be more sensitive to toxins known to target the mitochondria than standard assays. Early reductions in oxygen uptake were also observed on treatment with the apoptosis inducer camptothecin and exposure to UV light. Cytochrome P450{u2019}s, a family of oxygen dependent enzymes, were also analysed. These results indicate that the probes and measurement formats developed during the course of this work are of high utility for assessing cellular responses to drug treatment and in the analysis of oxygen-dependent enzymes and processes.
This book describes the methods of analysis and determination of oxidants and oxidative stress in biological systems. Reviews and protocols on select methods of analysis of ROS, RNS, oxygen, redox status, and oxidative stress in biological systems are described in detail. It is an essential resource for both novices and experts in the field of oxidant and oxidative stress biology.
In this thesis, the potential of phosphorescent metalloporphyrins with respect to development of biosensors and sensitive bioassays is discussed. In the first case, the broad area of fluorescent probes is discussed, with subsequent emphasis given to the area of time-resolved fluorescence. Particular reference is given to the state of the art of time-resolved probes, such as lanthanide chelates and cryptates, and their applications to both heterogeneous and homogeneous time-resolved fluorescence immunossays (TR-FIA). The potential of hydrophilic porphyrins is then discussed as a alternative to the conventional systems. Experimentally, p-isothiocyanatophenyl-derivatives of Pt(II)- and Pd(II)-coproporphyrin-I are described as stable monofunctional reagents which enable simple covalent labeling of proteins and other biomolecules under mild conditions in aqueous solutions. The bioconjugates, synthesised by optimised standard protocols, were characterized and subsequently used in solid-phase time-resolved phosphorescence immunoassays (TR-PIA) employing commercial time-resolved phosphorescence readers Victor2 and Arcus-1230 (Wallac). In the second section, the area of optical oxygen sensing is discussed with particular reference to measurement principles, sensor design including sensor configuration, dyes employed, encapsulation media, solid supports and instrumentation, and applications to date. The potential of metalloporphyrins as probes for optical oxygen sensing is discussed, concentrating subsequently on hydrophobic solid-state probes used in fibre-optic based measurement systems employing phase measurements. Experimentally, a cell viability assay based on the monitoring of respiration profiles of fission yeast by optical oxygen sensing is described a lifetime-based, non-invasive alternative to conventional viability assays. The system employs solid-state probe inserts, based on platinum octaethyl porphyrin ketone (PtOEPK), in a micro-titre format to measure respiration profiles and both chemical and biological effectors of cell growth. Also detailed is the development and characterization of a previously described reagentless enzymatic glucose biosensor using a phase-fluorometric oxygen transducer.
Research on dendrimers has exploded in the last 15 years, moving from the establishment of synthetic methodologies, particularly in the early years up to the end of nineties, towards sophisticated and wide-ranging applications. Dendrimers play an important role in many different areas, spanning from basic synthetic approaches to artificial photosynthesis, to medicine, to catalysis. The great potential of dendrimers is well-recognized by the hundreds of papers in the field and the increasing number of patents, and stimulated developments in other areas of knowledge, including new characterization techniques. However, some basic principles and methods still continue to give a unity to the field. Although several books on dendrimers have been published during these 15 years, the very recent progresses in new areas now requires a new point of view, trying to give a unifying and comprehensive outlook of the field. Since the first dendrimer was synthesized by Vögtle in 1978, dendrimers have experienced an explosion of scientific interest because of their unique molecular architecture. This resulted in over 5,000 scientific papers and patents published by the end of 2005. The proposed book will cover both fundamental and applicative aspects of dendrimer research. Chapters devoted to basic principles, synthetic methods and strategies, and advanced characterization techniques will be integrated by chapters illustrating the full potential of dendrimers in various fields, like artificial photosynthesis, multi-redox pool systems, diagnostics, biomedical and sensing purposes, design of functional nanostructures. Particular emphasis will be devoted to possible future developments.
This presentation describes various aspects of the regulation of tissue oxygenation, including the roles of the circulatory system, respiratory system, and blood, the carrier of oxygen within these components of the cardiorespiratory system. The respiratory system takes oxygen from the atmosphere and transports it by diffusion from the air in the alveoli to the blood flowing through the pulmonary capillaries. The cardiovascular system then moves the oxygenated blood from the heart to the microcirculation of the various organs by convection, where oxygen is released from hemoglobin in the red blood cells and moves to the parenchymal cells of each tissue by diffusion. Oxygen that has diffused into cells is then utilized in the mitochondria to produce adenosine triphosphate (ATP), the energy currency of all cells. The mitochondria are able to produce ATP until the oxygen tension or PO2 on the cell surface falls to a critical level of about 4–5 mm Hg. Thus, in order to meet the energetic needs of cells, it is important to maintain a continuous supply of oxygen to the mitochondria at or above the critical PO2 . In order to accomplish this desired outcome, the cardiorespiratory system, including the blood, must be capable of regulation to ensure survival of all tissues under a wide range of circumstances. The purpose of this presentation is to provide basic information about the operation and regulation of the cardiovascular and respiratory systems, as well as the properties of the blood and parenchymal cells, so that a fundamental understanding of the regulation of tissue oxygenation is achieved.
Oxygen is required for cellular respiration by all complex life making it a key metabolic profiling factor in biological systems. Tumors are defined by hypoxia (low pO2), which has been shown to influence response to radiation therapy and chemotheraphy. However, very little is known about spatio-temporal changes in P0 2 during tumor progression and therapy. To fully characterize and probe the tumor microenvironment, new tools are needed to quantitatively assess the microanatonical and physiological changes occurring during tumor growth and treatment. This thesis explores the design and construction of new oxygen sensors as tools for monitoring the tumor microenvironment in real-time. Semiconductor nanocrystals or quantum dots (QDs) are the basis of these tools. Previously, most imaging applications of QDs have used them as indicators of position; they have lacked a response to their local environment. Tethering a phosphorescent complex to a QD enables fluorescence resonance energy transfer to be exploited as a signal transduction mechanism, sensitizing the QD to oxygen. The mechanism for oxygen sensing involves kinetic quenching of the emission of the energy accepting phosphor in the presence of oxygen, while the emission of the energy donating QD remains stable. This mechanism was chosen owing to the unique ability of oxygen to quench emission from a phosphorescent compound, but not fluorescence from a QD. Phosphors such as osmium polypyridines (Chapter 2), Pd or Pt porphyrins (Chapters 3 and 4), or phosphorescent proteins (Chapters 5 and 6) may all be employed. An additional benefit of FRET excitation includes very large one- and two-photon excitation cross-sections of QDs. Together, these properties make the probes ideal candidates for 02 sensing applications in biological microenvironments, where probe concentrations may vary, and where the use of multiphoton excitation in microscopy presents significant advantages in imaging thick samples and in limiting extraneous tissue damage.