Application Of Fluorescent Probe In Molecular Detection And Cellular Imaging

Molecular imaging is the qualitative and quantitative study of biological processes in vivo,tissue,cell and molecule levels by using imaging methods.In recent years,a very popular frontier of scientific research has been widely used in early diagnosis of tumors,cell signaling,disease mechanism research,drug research and development,drug delivery,pharmacodynamic evaluation,and supramolecular hydrogel materials,and so on.Molecular imaging technology developed in recent years including Radionuclide Imaging(Single-photon Emission Computed Tomography SPECT and Positron Emission Tomography PET),Magnetic Resonance Imaging(MRI),Computed Tomography(CT),Ultrasound Imaging(USI),Optical Imaging(Bioluminescence Imaging BLI,Chemiluminescence Imaging,CLI,Fluorescence Imaging FLI),Photoacoustic Imaging(PAI).Those imaging techniques have their own characteristics and advantages in sensitivity,temporal and spatial resolution and tissue penetration.However,due to many factors togather,there are few successful molecular imaging trials for clinical diagnosis,which still remain a great challenge.Based on the development of molecular imaging technology mentioned above,we designed and synthesized several small molecule probes for high selectivity and high sensitivity detection of important biological molecules(reactive oxygen species,proteinases,metal ions,etc.)and used for intracellular and extracellular imaging analysis.This paper mainly includes three jobs as follow:Nitric oxide(NO)is the first ubiquitous signaling molecule in the human body.The selective and sensitive detection of NO in vitro and in vivo is of high importance but remains challenging.Previous fluorescent probes for NO detection either are of poor water solubility or lack selectivity over intracellular biomolecules.Herein,we rationally designed a water-soluble,biocompatible,small molecular probe o-phenylenediamine-Phe-Phe-OH(1)for the highly selective and sensitive detection of NO in vitro and in living cells.1 can react with NO and turn on the fluorescence emission at 367 nm via an ICT mechanism.In vitro tests indicated that 1 showed high selectivity for NO detection without interference from common anions,ROS/RNS,and intracellular biomolecules DHA,AA,or MGO.In PBS buffer,1 was applied for detecting NO within the range of 0-12 ?M with a LOD of 6 nM.Moreover,1 was successfully applied to sense intracellularly generated NO in living cells.The aggregation-induced emission(AIE)effect has recently been widely applied for biomarker sensing.But developing"smart"strategies to effectively aggregate the AIE fluorogen and additionally enhance the fluorescence emission remain challenging.In this work,by integrating a biocompatible condensation reaction with an AIE fluorogen,we rationally designed a "smart" dual AIE probe Ac-ArgVal-Arg-Arg Cys(StBu)-Lys(TPE)-CBT(1)for enhanced fluorescence sensing furin activity in vitro and in living cells.Compared with the single AIE probe Ac-Arg-Val-Arg-Arg-Lys(TPE)-OH(1-CtrI)which also subjects to furin cleavage,fluorescence emissions of probe 1 were additionally enhanced 1.7 fold and 3.4 fold in vitro and in living cells,respectively.We envision that,in the near future,our"smart" strategy of enzyme-instructed dual AIE could be widely applied for sensing(or imaging)enzyme activity in vitro and even in vivo with dramatically enhanced sensitivity.Calcium(Ca2+)as one of the most important ions of cells plays a critical role in electrical activities and biological processes.For the complex environment inside the cell,it is difficult to measure the concentration of Ca2+ of cells in vivo.NMR might be a practicable tool to gain the real-time information of the components in cells.Herein,we developed a novel Ca2+ sensitive and cell-permeable 19F probe 5TFM-BAPTA which can bind Ca2+by chelating with BAPTA.The chemical shift of 19F-NMR will change after binding with Ca2+ so that we can distinguish the different states of the probe with different concentrations of Ca2+in vitro.This probe penetrate the plasma membrane in the form of ethyl ester which can be cleaved in cytoplasm and allow the agent to bind Ca0·.This ethyl ester can also prevent extracellular Ca2+ binding.With the cleaved ester in the cytoplasm,we can semi-quantify the Ca2+ in cell by comparing the chemical shift of 19F spectrum with the fitting curve.It Vorks when it is used to measure the concentration of Ca2+ in cell lysis too.In cells,we find two different concentrations of Ca2+.After the further analysis,we speculated that the low concentration may represent the Ca2+ in the cytoplasm and the high one is because of the high concentration of Ca2+ in the endoplasmic reticulum(ER).For the phenomenon,the calcium-sensitive probe is supposed to distinguish the different levels of concentration of Ca2+ in the different organelles.