Construction And Application Of Ratiometric Flurescent Probe For Cu~+ And PH

The fluorescence probe can be applied in the detection and analysis of the biological inorganic species owing to its high-sensitivity,high-selectivity and real-time,reversible,in situ detection.The probe is divided into turn-on/off probe and ratiometric fluorescence probe.The latter overcomes the interference of environment and work concentration,providing the quantitative information.The usual strategy to design ratiometric probe is ICT,FRET and AIE.This thesis mainly designed ratiometric probe for Cu+and pH according to FRET and AIE mechanism.The labile copper pool can be used to maintain the copper homeostasis.The abnormal Cu+ level would catalyze Fenton reaction and generate reactive oxygen species,damaging the cells severely and irreversibly.Thus,the Cu+level is associated with Wilson disease,Menkes disease and cancer,etc.Currently,the reported fluorescence probes for Cu+were turn-on sensing probe and short-wavelength excitation probe,and they lacked of targeting to the organelles.Therefore,we wanted to design a two-photon and ratiometric,endoplasmic reticulum(ER)targeting probe for Cu+.We chose coumarin and benzooxadiazole as FRET donor and acceptor to construct a ratiometric probe.The coumarin could endow the two-photon ability and the sulfamide group generated the ER targeting ability.We designed and synthesized the probe CNSB.In the buffer,CNSB realized the quantitative detection of Cu+reversibly,overcoming the interference of the environment.Owing to the excellent biocompatibly and membrane penetration,CNSB was applied in the cell confocal imaging experiment.The experiment exhibited that CNSB could imaging the variation of the exotic Cu+ratiometrically and reversibly.Then CNSB was used to detect the variation of Cu+after incubating with cisplatin.The decreasing level confirmed the conclusion coming from the reported literature that cisplatin enterd cancer cells through CTR1,the transporting protein of Cu+.And the probe was proved to target the ER according to the colocalization experiments.So CNSB was used to detect the Cu+level during ER stress.CNSB successfully imaged the spatial and temporal variation of Cu+,providing the novel tool for physiological process about ER.In addition,Cu+level in the tissue was ugently needed to know except cells.And the probe wass proved to have the two-photon ability according to the large two-photon absorbance cross section.At last,because of the advantage of the two-photon imaging,CNSB detect Cu+level in the mice heart tissue slice.The probe could penetrate 60 μm,helping to detect exogenous Cu+in vivo.The suitable pH value is indispensable for the maintenance of intracellular physiological process,so cellular and subcellular pH benefits the cellular physiological balance.Owing to the abnormal environmental pH,the balance would be damaged irreversibly,leading to the cellular dysfunction.In addition,abnormal pH has been reported to be associated with several diseases.Thus,to develop novel ratiometric strategy to monitor pH will provide new idea for the development of tools for pH.In this thesis,naphthalene imide and phenyl group were linked with a single bond.The introduction of the morpholine and amino group resulted in the formation of intermolecular hydrogen bond,which prevented π-π stacking and leaded to aggregation induced emission.The protonation in the acidic environment broke the hydrogen bond and induces deaggregation,so the aggregation fluorescence decreased accompanied by the enhancement of the intrinsic fluorescence,resulting in the quantitative detection.We obtained four molecules by combining the morpholine and amino group with the fluophore.The emission spectra,DLS and TEM experiment demonstrated that the AIE characteristic of four probe.And the aggregation emission appeared in the range of 550-600 nm,accompanied by the nanoaggregation of the probe in the poor solvent.The crystal structure of EtPPN(CH3)2 proved the existence of intermolecular hydrogen bond and our hypthesis.The data confirmed that the red-shift emission of the probe came from the morpholine group.In acidic environment,the protonation of pH responding group broke the hydrogen bond,helping the ratiometric detection of pH.What’s more,the detecting behavior was high-selective and reversible.The pH detecting range was controlled by the combination of different pH responding group.The work showed that the modification of the structure helped to modify the aggregation of the molecule and affect pH detecting range,which contributed to the new strategy of pH quantitive detection.