| The intracellular redox balance plays an important role in maintaining the normal function of living organisms.Intracellular reactive oxygen species?ROS?are closely related to various biological processes such as intracellular redox balance and signalling transduction.Excessive production of ROS could lead to the occurrence and development of diseases.Superoxide anion radical(O2·-)is the first ROS produced in cells and is an important regulator factor.Excessive production of O2·-induces apoptosis of cells and the occurrence of various diseases,such as neurodegenerative diseases and ischemia reperfusion injury.Studies have shown that O2·-exists in multiple organelles and plays a different and crucial role in the regulation of cell functions.Therefore,it is helpful to exploring the dynamic changes of O2·-level in various organelles on the signaling pathways of related diseases,so as to solve the mystery of the molecular mechanism of various diseases.However,the current development method for the O2·-detection does not have the ability to dynamically,reversibly and real time analyze of O2·-in each organelle.Hence,it is necessary to develop powerful tools to achieve real-time detection of O2·-fluctuations in organelles.The two-photon fluorescence imaging method has the advantages of high sensitivity,deep penetration depth,high spatial and temporal resolution,etc.It is an important means to analyze the changes of active small molecules in organelles.In recent years,many molecular probes with two-photon fluorescence properties have been developed to realize the visualization of intracellular O2·-,but there are fewer molecular probes that possess both organelle targeting and dynamic reversible detection of O2·-properties.It also limits revealing the relative signaling pathways mediated by O2·-.Therefore,it is necessary to design and prepare two-photon fluorescent probes that can target the organelle and dynamically reversibly detect O2·-.The main contents of this paper are as follows:1.Design and synthesize a two-photon fluorescent probe CCA that targets the Golgi and dynamically reversibly detects O2·-.L-cysteine was selected as an anchoring group for the Golgi,and a caffeic acid ester which specifically reacts with O2·-as a recognition group.CCA is capable of detecting O2·-with high selectivity,sensitivity,transients,and reversibility.CCA could detect O2·-linearly in the range of O2·-concentration 0-5?M.The linear equation is F=1621.51[O2·-]??M?+631.98,the linear correlation coefficient is 0.991,and the detection limit is 18 nM.CCA is capable of imaging O2·-fluctuations in Golgi apparatus and enabling deep imaging of the mouse's abdomen and liver,with a depth of penetration of 350?m.With assistanceoftwo-photonfluorescenceimagingofhepatocytesandmiceafter ischemia-reperfusion?IR?,we find that overproduction of Golgi O2·-during the hepatic ischemia-reperfusion injury can activate caspase2 and lead to apoptosis.Besides,the level of O2·-in Golgi is positively regulated by tumor necrosis factor?TNF-??.The experimental results reveale the signaling pathway of O2·-in the Golgi process in the liver of IR mice,and elucidate the molecular mechanism of O2·-mediated IR injury,providing a new strategy for the treatment of liver IR injury.2.Design and synthesize a two-photon fluorescent probe CA-DPPE that targets the cell membrane and detects the change of O2·-level instantaneously.CA-DPPE employs1,2-dipalmitoyl-sn-propyltriyl-3-and diethylaminophosphate?DPPE?as targeting groups for cell membranes,and caffeic acid as an O2·-specific recognition group.CA-DPPE has a high selectivity and reversibility towards to O2·-.Utilizing two-photon fluorescence imaging,we successfully detected changes in O2·-concentration on the cell membrane. |
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