Design And Synthesis Of Fluorescent Probe Based On ICT And Its Application In Biological Imaging

Fluorescence detection techniques of fluorescent probe have been widely used in areas including chemistry,materials science and biology due to its simple operation,high sensitivity,specific selectivity,low cytotoxicity and low detection limit.Mitochondria,a well-known energy supplying organelle in almost all eukaryotic cells,play vital roles in cellular metabolism.The concentration and distribution of various active analytes in mitochondria,such as reactive oxygen species（ROS）and metal ions,as well as microenvironment,such as p H value,temperature,polarity,hypoxia and viscosity,etc,are closely related to the physiological functions of mitochondria.Mitochondrial targeting probe could play significant roles in the early diagnosis of diseases related to mitochondria.Therefore,the development of highly sensitive fluorescent probes for real-time in-situ,dynamic,accurate and reversible monitoring the various analytes in mitochondrial is significant.In this paper,we synthesized three kinds of probes for the detection of hydrazine（N₂H₄）,hypochlorite（Cl O^-）/carboxylesterase（CEs）,CEs,respectively.The main contents are as follows:（1）Hydrazine（N₂H₄）is carcinogenic,extremely toxic,and induces serious environmental contamination and physiological dysfunction,however,it is widely used as an industrial material.In this work,an intramolecular charge transfer（ICT）-based fluorescent probe,（Z）-1-（4-acetoxybenzyl）-4-（1-cyano-2-（7-（diethylamino）-2-oxo-2H-chromen-3-yl）vinyl）pyridin-1-ium named probe 1,was designed for the dual-excitation（420 and 600 nm,excitation separations>160 nm）,near infrared（NIR）-emissive,and ratiometric fluorescent detection of N₂H₄.The sensing behavior of probe1 for N₂H₄ detection was available over a wide p H range,and detection limits of 60 n M and 2 n M were achieved at excitation 420 and 600 nm wavelengths,respectively.In addition,probe 1 was successfully used to image mitochondrial N₂H₄ in living cells and zebrafish.And the probe was also successfully applied to image the N₂H₄ signal in test strips and soils.（2）The detection of hypochlorite（Cl O^-）and carboxylesterases（CEs）in vivo is of great importance to comprehend the accurate effect of Cl O^-/CEs in various pathological and physiological processes.We herein presented a hydrolysis-based fluorescent probe2 with dual response sites and ratiometric response to CEs,and the probe can also be oxidated by Cl O^-as an on-off fluorescent probe.Probe 2 could detect CEs and Cl O^-with excellent selectivity and sensitivity(with low detection limit of 1.1×10^-6 U/m L and 65 nΜ).Interestingly,this probe was found to be applicable in a broad p H range（4.0-13.0）.Additionally,as well as the accompanied by fluorescence transformation form blue turns into green with addition of CEs.The sensing mechanism was carefully confirmed by the electrospray ionization mass spectrometry（ESI-MS）analysis,¹H NMR studies and density functional theory（DFT）calculations.Significantly,due to the pyridinium targeting,the probe 2 were successfully applied to bioimaging CEs/Cl O^-in living cells mitochondria and zebrafish.Therefore,probe 2 is an effectively tool for visualizing Cl O^-/CEs at the cellular and subcellular levels.（3）Carboxylesterases（CEs）are widely distributed in the body and they are closely associated with their important roles in drug metabolism and the abnormal levels often cause various diseases such as cancers.Herein,we proposed a new design strategy of fluorescence-electrochemistry combining,which led to the synthesis of a new fluorescent probe（termed as probe 3）with high specificity toward both CEs and improved sensitivity.In our design,ferrocene unit not only serves as a part of probe 3structure,but also affords electrochemical signals.The probe showed high selectivity to CEs and a“Turn-On”fluorescence response,which can be utilized for detection of CEs and imaging in complicated biological samples including living cells and zebrafish.Furthermore,the hydrolysis of the acetyl moiety by CEs of the probe 3,resulting in the decrease of electrochemical response and the increase of fluorescence signal.All of these findings suggest that probe 3 is the well-designed multimodule probe for real-time tracking and imaging endogenous CEs in complicated biological samples.