New Type Of Hypoxia Fluorescent Probe: Design, Synthesis And Application

There were various micro or trace amounts of active species in living systems. Cells are thebasic unit of life activities and require a variety of substances in their metabolic process.Molecular oxygen is one of paramount importance for the functioning of aerobic cells andorganisms. Once oxygen concentration is out of normal range, cell functions of influence isunavoidable, induced a series of related diseases. So, qualitative and quantitative analysis ofoxygen is significantly meaningful in theory and practice. Fluorescent method has manyadvantages, such as high sensitivity and selectivity, especially in chemistry, biology andmedicine research fields. What is more inviting is that fluorescent probes can penetrate intoliving cells with little injury, and interaction with dissolved oxygen (DO) to form stronglyfluorescent substance. With the help of confocal laser scanning microscopy imaging, on-line realtime visualization detecting of cellular oxygen can be achieved. Based on above-mentionedreasons, the probe conceivably is suited for living applications. Hypoxia is a common feature ofvarious diseases. Therefore, hypoxia-specific molecular probes would be useful as fundamentalresearch and diagnostic agents. Consequence, development of superior oxygen indicator hasgreat significance and necessity.Various shortages exist in the present methods. Thereinto, sensors base on luminescence quenchby molecular oxygen. Although, they are long excited state lifetime, large Stokes shift, and so on,high activity of singlet oxygen generate in reaction process. However，fluorescent probe basedon redox mechanism generate hydrogen peroxide and hydroxyl radical. Reactive oxygen species(ROS) oxidize amino acid, protein, DNA, modify structure and functions, effect cell signaltransduction, cytotoxicity manifestations, and even kill cell. Consequently, design and synthesisprobes which don’t produce singlet oxygen. Beyond question, the measurement and imaging ofoxygen levels in live cells and tissue supply powerful instrument in modern biology, physiology,and medicine. The main contents of this thesis were shown as follows:In chapter one, we have summarized research significance and research status of oxygen andhypoxia. In chapter two, we designed and synthesized a new NIR fluorescent probe,2-(2-nitro-1H-imidazolyl) ethylamine as specific center, tricarbocyanine as fluorescent dye, withhigh selectivity and high sensitivity. The probe effectively avoided the influence ofauto-fluorescence from biological samples and modulated the fluorescence emission intensitythrough conversion between nitro and amino. Nitro was selectively and rapidly reduced at roomtemperature to amine in good yields by employing hydrazinium monoformate, in the presence ofcommercial zinc dust and hypoxia. And Nitro was selectively and rapidly reduced atphysiological conditions by employing nitroreductase in the presence ofβ-Nicotinamide adeninedinucleotide and hypoxia. Then fluorescence of probe was recovered. Therefore, hypoxia couldbe detected. The fluorescence increase is proportional to the concentration of hydraziniummonoformate in the range of 0-10.3μM, linear equation is F = 49.77634 + 157.60492[Formylhydrazine] (μM), R = 0.9973. The limit of detection is 10. 81 nM. Besides, thefluorimetric experiments of the probe showed that the sensitive fluorogenic reagent features ahigh selectivity over other biological reductants: ascorbic acid, glutathione reduced, L-cysteine.Confocal microscopic imaging of hypoxia drived from hepatocellular carcinoma cell revealedthat the probe is lower toxicity, good water solubility and biological compatible.