| Many kinds of free radical have been found in metabolic processes. They play important roles in maintaining normal physiological function of organism, meanwhile they are also closely related to body ageing and various diseases. Their dramatic increase can break metabolic balance in vivo, and result in physiological function disorders. Ozone (O3) is a member of reactive oxygen species (ROS), stratospheric O3 prevents mass solar ultraviolet ray from reaching the earth's surface, while troposphere O3 is endangering safety of biosphere. Endogenous O3 is easily induced when the body responses to inflammation or external stimulation, which can lead to abnormity of cellular function by changing plasma membrane, affecting ionic channels, decreasing enzyme activity etc., and even cause several diseases (skin cancer, respiratory diseases, et al.). Presently, only several methods have been advanced for O3 detection, such as chemiluminescence method (CL), fluororesence method, etc. Due to lower sensitivity, lower selectivity and longer reaction time, there is not too much research on O3 as on other ROS. To date, the physiological function of intracellular O3, signal transduction pathways engaging O3, relationship between O3 and disease are still unclear, so development of O3 detection methods suitable for biological systems is of great study significance.When fluorescence probe reacts with intracellular free radical, the fluorescence intensity or absorption wavelength of it can change, then it is able to visually detect the active small moleculars in living cells and real time supervise their dynamic changes in vivo. Based on this mechanism, the aspects of investigation are as follows:A new near-infrared (NIR) fluorescent probe (Trp-Cy) for ozone detection was designed and synthesized in this work. Reaction conditions of Trp-Cy with O3 were optimized in chemical systems. The relative fluorescence intensity of Trp-Cy responsing to H2O2(120μM), NaC10 (24μM),·OH, t-BuOOH (30μM), GSH and Vc (300μM) were quite low, which suggested they have no interference with O3 detection. Trp-Cy can respond to O3 instantly, with a significant increasing of fluorescence intensity, then reached the peak and kept stable. Fluorescence intensity of Trp-Cy can be significantly enchanced with the ozone concentration, the linear equation was F= 10341.7+1486.51394×[O3] ([O3]:μM), R= 0.99847. O3 concentration ranging from 1.0 X 10-7 to 7.0 X 10-6M. It demonstrated Trp-Cy can detect weak changes of O3 concentration. MTT assay in vitro was carried out to study effects of Trp-Cy on cell system. When the cell survival fraction was 50%, the probe concentration was 465 uM, which suggested Trp-Cy is low toxicity to cells. Real time detection of O3 in lung cancer A549 cells was achieved using confocal laser scanning microscope.As stated previously, Trp-Cy has the following characteristics:(1) the maximum excitation and emission wavelengths are in near-infrared (NIR) region (at 630 nm,770 nm, respectively), which could effectively avoid background fluorescence interference in cells, and alleviate cells damage; (2) it has a larger stokes shift (up to 140 nm), which can avoid self-quenching effectively and improve the detection sensitivity and accuracy; (3) it reacts swift with O3, not being affected by other active moleculars or metal ions existed simultaneously, which is helpful to real time detect fractional changes of intracellular O3 concentration; (4) its autofluorescence intensity is weaker, but increases significantly when combined with O3; (5) it can target mitochondria specifically.The fluorescent probe with high sensitivity, swift response and lower toxicity will play an important role in study of effects of O3 on biological systems. |
PDF Full Text Request