Design Of Novel Functional Fluorescent Probes For Imaging Analysis Of Bioactive Species Under Mercury Stress

Fluorescence imaging analysis technology has advantages of high sensitivity as well as good selectivity,and it can realize the dynamic detection of targets in real-time and in situ.Thus,fluorescence imaging analysis technology has gradually become an indispensable imaging analysis tool in the fields of environmental analysis and biological analysis.In this dissertation,we focus on the construction of novel fluorescent probe tools based on fluorescence imaging analysis technology for imaging and monitoring of endogenous biologically active species under mercury stress.The impacts of mercury stress on human health can be clarified by studying the distribution,concentration changes,transport and other biological effects of bioactive species on the levels of cells,tissues and in vivo,which will provide technical support and scientific basis for further studying the mechanism of mercury poisoning.The details of this dissertation are as follows:1.A three-channel fluorescent probe for associated-detection of superoxide anion and mercury ion in mercury poisoning modelsAs a cytotoxic heavy metal ion,mercury ions(Hg²⁺)induce severe oxidative stress and further result in physiological dysfunction.In order to explore the complex mechanism of mercury poisoning,a three-channel ratiometric fluorescent probe HCy-Se H for associated-detection of superoxide anion(O₂^·-)and Hg²⁺is designed and synthesized.Fluorescence response of the probe initiates by hydrogen abstraction reaction from the hydrocyanine fluorophore moiety.Probe HCy-Se H recovers its?-conjugated system and converts to heptamethine cyanine derivative Cy-Se H once oxidized by O₂^·-.Cy-Se H coexists with its conjugate base Cy=Se.The response unit selenol can trap Hg²⁺via selenium-mercury antagonism reaction to afford final product Keto-Cy accompanying the change in fluorescence emission wavelength.The probe offers high selectivity and sensitivity towards O₂^·-and Hg²⁺,and the probe can be applied for associated-detection of O₂^·-as well as Hg²⁺.The probe is applied for O₂^·-and Hg²⁺detection in mercury poisoning cells and mice models.And the experimental results demonstrate that mercury poisoning can cause severe oxidative stress and induce kidney tissue damage.Thus,the probe contributes to revealing the pathological mechanisms related to mercury poisoning.2.A sulfydryl-based near-infrared ratiometic fluorescent probe for comparative study of acute and chronic mercury poisoningOur previous work,a three-channel fluorescent probe for O₂^·-and Hg²⁺ associated-detection,has aroused great concern.Given the complex pathological mechanism of mercury poisoning,we hope to further reveal the difference between acute mercury poisoning and chronic mercury poisoning for better diagnosis and treatment.However,the stability of probe in the previous work does not meet our requirement.Based on the great advantages of the three-channel ratiometric fluorescent probes,heptamethine cyanine dye is selected as fluorophore and thiol group is chosen as response group to construct a more stable fluorescent probe Hcy-SH for mercury poisoning detection.The probe shows good spectral characteristics towards O₂^·-and Hg²⁺.A comparative study of acute mercury poisoning and chronic mercury poisoning is conducted by the probe,and results demonstrate that mercury poisoning can cause severe O₂^·-outbreaks.Outbreaks of O₂^·-in acute mercury poisoning are much more severe than chronic mercury poisoning,especially in the heart.Unlike acute mercury poisoning,the outbreak of O₂^·-in mouse models of chronic mercury poisoning varies in different organs and different times.However,regardless of acute or chronic mercury poisoning,Hg²⁺is mainly accumulated in kidney.The experimental results show that the probe HCy-SH is a potential candidate for accurate diagnosis and evaluation of mercury poisoning.3.Ratiometric fluorescent probe for detection of glutathione hydropersulfides under mercury stressGlutathione hydropersulfides(GSSH)are alluding to play crucial roles in signal transduction,redox homeostasis,and metabolic regulation.However,the detailed biological functions of GSSH in these aspects are extremely ambiguous.The key barrier to studying GSSH in biological systems is a lack of detection tools with high spatiotemporal resolution.To address the issues,we design a novel fluorescent probe TP-Dise to realize GSSH detection in living cells and tissues.Using probe TP-Dise,the biosynthesis of GSSH is investigated and the results indicate that GSSH are mainly from two sulfurtransferases,cystathionine?-synthase and cystathionine?-lyase,which are consistent with previous research reports.Furthermore,the biological functions of GSSH in protecting cells from Hg²⁺induced cell damage are explored for the first time.The results of the apoptosis experiments demonstrate that GSSH can alleviate the mercury stress damage and protect cells.Probe TP-Dise is a powerful candidate for evaluating the physiological pathology functions of GSSH,which will promote research on the biological function of GSSH.4.Glutathione peroxidase-activatable ratiometric fluorescent probe for imaging analysisGlutathione peroxidase(Gpx)is one of the vital proteases in the first-line antioxidant defense system and it participates in regulation of redox homeostasis as well as the pentose phosphate pathway.However,the current methods cannot achieve in situ visualization studies of Gpx.In addition,Gpx is highly reactive and susceptible to external interference.Herein,a novel ratiometric fluorescent probe(TP-SS)for Gpx detection is constructed.Using TP-SS,we explore the reversible catalytic cycle and the antioxidant mechanisms of glutathione peroxidase/glutathione(Gpx/GSH)redox pool.The fluctuations of Gpx in aging cells and mice models are investigated,and the results demonstrate that the activity of Gpx is decreased during aging.The antioxidant mechanism of Gpx in mercury poisoning cell and mice models are also studied.The results illustrate that the concentration of Gpx is decreased by Hg²⁺,and Hg²⁺further results in oxidative stress as well as activates apoptotic pathway through inducing reactive oxygen species burst,thereby aggravating the organism damage.Gpx/GSH redox pool can effectively relieve oxidative stress using GSH as the obligate cosubstrate to reduce reactive oxygen species.The concentration fluctuation of Gpx in deep brain tissues is further studied by the probe.The novel probe is a promising chemical tool for exploring the physiological functions of Gpx and the cellular redox homeostasis events,and we expect that the novel ratiometric fluorescent probe can facilitate the development of Gpx-targeting tools as well as offer great advances in exploring the physiological and pathological functions of Gpx.5.A ratiometric fluorescent probe for bioimaging analysis of endogenous biological signaling molecule superoxide anionBioactive species play vital physiological functions against various environmental pollutions such as mercury pollution.However,the physiological and pathological functions of these bioactive species in other aspects are also worth further exploration.Taking O₂^·-as an example,mercury poisoning can cause severe O₂^·-burst.However,high concentration of O₂^·-can activate the tumor apoptosis signaling pathway and effectively kill tumor cells.Here,we design and synthesize a probe tool TP-Tfs for visual monitoring of endogenous biological signal molecule O₂^·-,and further study its physiological functions in other aspects.The probe shows good selectivity,and it can realize the rapid detection of O₂^·-as well as O₂^·-analysis in mercury poisoning cells.In addition,the probe is employed for endogenous O₂^·-detection during tumor treatment in cells and tumor-bearing mouse models.The experimental results show that the chemotherapeutic drug cisplatin can induce an increase in intracellular O₂^·-concentration and effectively kill tumor cells.Curcumin combines with the chemotherapeutic drug cisplatin can induce more serious O₂^·-burst and curcumin can sensitize the chemotherapeutic drug cisplatin,thereby improving the therapeutic effect of tumors.Therefore,the physiological functions of O₂^·-are worth exploring in mercury poisoning and other aspects,such as tumors.The probe TP-Tfs plays apotential roles in drug screening and tumor treatment evaluation.