| Intracellular pH plays critical roles in various biological processes, such as cell proliferation and apoptosis, ion transport, endocytosis, and exocytosis, etc. Some organelles, such as endosomes and plant vacuoles, have pH in the range of 4?6. The acidic environments in lysosomes?pH 4.5?5.5? can facilitate the degradation of proteins. Normally, cell dysfunction is closely related to abnormal intracellular pH value, which is associated with some major diseases, such as cancers and Alzheimer's. Therefore, the efficient monitoring of intracellular pH is helpful to the study and understanding of physiological and pathological processes of living organism, which has aroused great concern of the researchers.Free radicals play an important role not only in many chronic diseases but also in some normal biological processes. Superoxide anion(O2·-), is the first radical that generated from mitochondria respiratory chain, possessing medium oxidizing ability and nucleophilicity. Nitric oxide?NO?, the known messenger molecule, can diffuse into biological membrane quickly and play important roles in vascular regulation, immunoregulation, and neuroregulation. It is well known that peroxynitrite?ONOO-?, a potent oxidant and nucleophile, is formed by the reaction of O2·- and NO. There is plenty of evidence show that ONOO- can attack and oxidize proteins, lipids, carbohydrates, and nucleic acid, resulting in irreversible injury to organism. Hence, the accurate detection of ONOO- is of great significance to explain the molecular mechanism relating to its toxicity.Fluorescent probe-based fluorescence imaging show the advantages in terms of non-destructivity, in situ visualization, and simple operation. Quantification of pH or ONOO- in living cell or animal may provide more accurate information than the qualitative results. However, since the inhomogeneity of biological samples, fluctuation of light source, variation of probe concentration, and probe leakage from cells, the fluorescent probes characterized of single excitation/single emission will unavoidably be affected by the above factors, giving untrue information. Fortunately, ratiometric probes can not only avoid the above factors, but also give quantitative information about the analytes. The accurate and reliable information drawn from complex biological samples is thus ensured.Based on the above consideration, two type of ratiometric fluorescent probes have been designed and synthesized and were used to detect pH and ONOO- in living cells and in vivo.1. In this section, utilizing a cyanine fluorophore framework, three NIR ratiometric probes featuring with an acceptor-donor-acceptor structure have been synthesized. With the increase of pH value, the fluorescence intensity of the emission at long wavelength decreased, while that at short wavelength increased. There are 240?260 nm difference in wavelength between the two emission peaks, exhibiting high resolution in emission spectra. Et-CY showed sensitive response to pH in an intracellular pH range of 4?8. Utilizing Et-CY, the pH changes of Hep G2 cells under hypoxia and during oxygen-glucose deprivation/reperfusion was quantified. In addition, pH variation in living mice was also detected by Et-CY. The present results have provided suitable pH probes with good resolution and wide responsive range to the field of bioanalysis.2. In this section, BODIPY and hemicyanins have been coupled to construct a new ratiometric fluorescent probe?BODIPY-Ratio? specific for ONOO-. ONOO- will readily oxidize and break the carbon-carbon double bond of BODIPY-Ratio, giving a ratiometric signal when the emission at long wavelength decreased, while that at short wavelength increased. BODIPY-Ratio showed satisfactory sensitivity and good selectivity toward ONOO- and had been used to image endogenous ONOO- in living cells. In addition, ONOO- in living mice was also detected by BODIPY-Ratio. The present results have provided suitable probes with fast and ratiometric response for ONOO- to the field of bioanalysis. |
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