| Free radicals are metabolizable offspring of manifold biochemistry reaction in living systems. Various reactive oxygen species (ROS) are generated in the course of biological metabolism, such as superoxide (O2-·), hydrogen peroxide (H2O2), hydroxyl radical (HO·), nitric oxide (NO) and peroxynirte (ONOO-), these ROS play a vital role in physiology. A rapid rise in intracellular oxidant levels under oxidative stress could cause damage to biological molecules and result in various diseases, such as carcinogenesis, inflammation, ischemia-reperfusion injury, and signal transduction. They are important mediators for the pathological conditions of various diseases. To date, several methods for O2-·detection include electron spin resonance spectroscopy, SOD-inhibitable nitroblue tetrazolium, chemiluminescence, and fluorescent method. However, the short live and low concentration of ROS mean to the determination difficult, especially the determination in vivo and in situ has no breakthrough. At present, Fluorescence imaging methods are sensitivity, selectivity, ease of use and takes full advantage of benefits provided by confocal laser scanning microscopy (CLSM). CLSM is a type of high-resolution fluorescence microscopy that can give the fluorescence image of cells and tissues, where ROS were detected by fluorescent probe. Fluorescence imaging method challenged the traditional fluorescent method and achieved the detecting of ROS in vivo and in situ. There is an exigent need for researchers to develop fast-respond probes to trap ROS for investigation of the mechanisms of the production, metabolism and trafficking. Therefore, considering the design applicable probes, it is of great importance to improve reaction rate and selectivity to avoid potential side reactions from other ROS under conditions that guarantee high sensitivity. If novel fluorescence probes that overcome the above problems were available, they would contribute greatly to the elucidation of the roles of ROS in living cells.In addition, anions present in the human body, play essential roles in many biological processes. Particularly, fluoride is indispensable trace element in creature vital activity. The effect of fluoride on humans has a dual role as an essential element and at high levels as a toxic substance. Excess fluoride ion results in fluorosis and renal, gastrointestinal and immunological toxicity. Thus, the process of quantifying trace fluoride anion within biological samples, elucidation of the complex physiological and pathological roles provide motivation for developing new ways to study dynamic fluoride chemistry in living systems. In the reported several types of molecular probes for fluoride detection, hydrogen bonding between the N-H of a urea or pyrrole group and fluoride was used for the recognition. On the other hand, there have been a few reports regarding fluoride ion detection utilizing a unique fluoride-boron interaction and fluoride- silica interaction. But, no small molecules have ever been available for detection and imaging fluoride in living biological systems.Metal ions present in the human body, play essential roles in many biological processes. Among them copper is an essential element in creature vital activity, fulfilling a dual biological role. Many cytosolic, mitochondrial, and vesicular oxygenprocessing enzymes require copper as a redox cofactor, but uncontrolled reactions of copper ions with oxygen and reactive oxygen species (ROS) can also trigger oxidative damage to proteins, nucleic acids, and lipids. Failure to maintain copper homeostasis in the body, organ or cell, may lead to erious neurodegenerative diseases, including Menkes and Wilson diseases, familial amyotropic lateral sclerosis, Alzheimer's disease, and prion diseases. In order to explore the mechanisms by which copper mediates various physiological and pathophysiological processes, the development of a method capable of detecting copper in biology has been an intriguing challenge for chemists, and biologists.Thus, effective probes for ROS, fluoride and copper ion should feature avorable compatibility with biological samples, low toxicity, and resistance to oxidation, visible-wavelength excitation and emission profiles to minimize sample damage and native cellular autofluorescence, and a turn-on or ratiometric fluorescence response for mapping spatial resolution.Based on the changes in spectrum characters of the fluorescent probes reacting with reactive oxygen species, fluoride, and copper ion, we have carried out three aspects of investigation:First, a spectrofluorimetric method using 2-chloro-1, 3-dibenzothiazoline cyclohexene as a new fluorescent probe was developed for the determination of superoxide anion radical (O2-·). It is a non-fluorescent compound, after treatment with O2-·, generates the fluorescent product. The new probe has high selectivity and sensitivity in detecting O2-·. The fluorescence images of probe-stained macrophages and stimulated with PMA were obtained successfully by using confocal laser scanning microscope. In addition, the determination of O2-·in cell extracts was performed to further confirm the feasibility of the proposed method in biological systems.Second, a spectrofluorimetric method using fluorescein Di-t-butyldimethylsilyl ether (FDSiE) as a new fluorescent probe was developed for the determination of fluoride in aqueous solutions. Upon treatment with F-, FDSiE, a closed and weak-fluorescent lactone, was transformed into an open and strong fluorescent product. The fluorescence images of probe-stained and F--supplemented macrophages were obtained successfully by using confocal laser scanning microscope.Third, this paper presented the synthesis and the properties of a new Near-Infrared fluorescent probe, Cy.7. hydrazide. |
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