Construction Of Hydrogen-peroxide-responsive Cyanine Fluorescence Probes And Their Application In The Detection And Imaging In Plants

Reactive oxygen species（ROS）are important bioactive substances in plants and other organisms,which play key roles in many biological processes.Therefore,it is important to detect and quantify ROS levels.Fluorescence imaging is a convenient and cost-effective method for bioanalysis and therapeutic monitoring that can be used to detect and quantify ROS levels.In fluorescence imaging,fluorescent emission in different wavelength regions can be used as the reporting signals,for example,the visible light wavelength range（400-700 nm）,the first near-infrared light wavelength range（NIR-I,700-900 nm）,and the second near-infrared light wavelength range（NIR-II,900-1700 nm）.Due to the shallow tissue penetration depth of visible light and the strong autofluorescence of tissues,the research on fluorescence imaging in recent years have mainly been focused on the near-infrared（NIR）region.NIR-II light has little photon scattering,low tissue absorption,and higher tissue penetration depth.Therefore,NIR-II fluorescence imaging has the advantages of significantly reduced background interference and being able to visualize biological analytes and tissue structures in deep tissues,and has made remarkable achievements in various biosensing and bioimaging applications.Although the research on NIR-II fluorescence imaging in animals has achieved great progress,in the study of plant fluorescence imaging,the emission light of the commonly used probes is in the visible region,and currently operable high-performance activatable NIR fluorescent probes are still very scarce.To solve the above problems,three NIR cyanine fluorescent probes with H₂O₂response have been designed and prepared in this paper,and their applications in fluorescence imaging in plants have been explored.The main contents are as follows:（1）The H₂O₂-responsive D-A type semi-cyanine fluorescent probe CTX-B based on cyanothiazole derivatives has been designed and synthesized.As for the probe,dioxaborolane as the biomarker recognition unit（response unit of H₂O₂）and fluorescence quencher is connected to the xanthene framework,and the electron donor part（D）and the cyanothiazole derivative as the electron acceptor part（A）are coupled by Knoevenagel condensation reaction to render the D-A semi-cyanine probe CTX-B.By establishing the model of oxidative stress in soybean sprouts induced by short-term metal ion pollution,the response of the probe CTX-B to H₂O₂ and its fluorescence imaging ability for oxidative stress in soybean sprouts have been investigated.With the model of oxidative stress induced by short-term metal ion pollution of Chinese cabbage seedlings being further established,the imaging and detection of oxidative stress in Chinese cabbage seedlings have been realized by using probe CTX-B.（2）The D’-D-A semi-cyanine fluorescent probe CT-XA-H₂O₂ based on cyanothiazole derivatives has been designed and synthesized,which is responsive to H₂O₂.First,the biphenylamine group is introduced into the xanthene molecular framework through the reaction of secondary amine and bromine,and the chromophore with aggregation induced emission characteristics is obtained.The dye CT-XA-OH is prepared by reacting this chromophore with cyanothiazole derivatives as electron acceptor,and its emission wavelength reaches 1036 nm.On the other hand,the H₂O₂ responsive unit（dioxborane）is introduced into the diphenylamine-xanthene molecular skeleton by substitution reaction.The probe CT-XA-H₂O₂ is synthesized via Knoevenagel condensation reaction between the electron donor（D’-D）and cyanothiazole derivatives（A）.The oxidative stress models of soybean sprouts and peanut sprouts induced by cadmium ion or high salt pollution have been employed to explore the response of CT-XA-H₂O₂ to the overexpression of H₂O₂ in soybean sprouts and peanut sprouts,and the imaging and detection ability of oxidative stress in soybean sprouts and peanut sprouts.（3）The cyanine fluorescent probe ITC-APA-H₂O₂ that can respond to H₂O₂ based on tert-butylcyclohexene is designed and synthesized.Heptamethine cyanine molecular skeleton is synthesized by indole salt and tert-butylcyclohexanone;and then through electrophilic reaction,acetyl and pyridine derivatives are connected with heptamethine cyanine skeleton to render the dye ITC-APA.ITC-APA-H₂O₂ is synthesized from ITC-APA and dioxaborolane（response unit for H₂O₂）.Due to the intramolecular photoinduced electron transfer（PET）effect,the fluorescence intensity of the probe（peak at 922 nm）is significantly reduced compared with the dye.The models of oxidative stress induced by indoleacetic acid（plant auxin）in rice seedlings and corn seedlings have been established to investigate the response of the probe to the overexpression of H₂O₂ in the process of oxidative stress in rice seedlings and corn seedlings,and the imaging ability of the probe to oxidative stress in rice seedlings and corn seedlings.