Design,Synthesis And Imaging Research Of Fluorescent Probes Based On Nile Red Fluorescence Platform

In recent years,fluorescent probes have become indispensable tools in modern science（materials chemistry,analytical and environmental chemistry,biochemistry）and medical domain（clinical diagnosis,biotechnology,molecular biology）.Fluorescence imaging technology is a kind of tracing method which can realize the subtle changes of corresponding objects to be measured in the course of disease,and it also has multifarious advantages including non-invasive,in-situ detection,visualization and high spatial and temporal resolution.As organic functional materials,organic fluorescent dyes are widely used in chemistry,physics,biology,environmental science,medicine and other fields.Meanwhile,the development of designing excellent fluorescent dyes in electroluminescent materials,photosensitizers,fluorescent probes have attracted more and more attention.Compared with inorganic fluorescent materials,organic fluorescent materials have numerous advantages,such as structurally modifiable,high luminous efficiency,wide emission range,good film forming property and so on.Therefore,organic luminescent functional materials have always been of interest to scholars,especially the dyes which emit near-infrared（650 nm-900 nm）fluorescence.Nile Red is a well-known lipophilic oxazine organic dye.Due to the long emission wavelength of Nile Red,it is also a remarkable backup dye for near infrared emission.Meanwhile,Nile Red has attracted much attention because of its excellent photophysical and photochemical properties.For example,large rigidπstructure,long emission wavelength,two-photon properties,good photophysical and chemical stability,good lipid solubility（strong fluorescence in many organic solvents）,wide operating wavelength range,high quantum yield（0.28 in Me OH）,fluorescence is not affected by p H 4.5-8.5,etc.Based on these above advantages,dye Nile Red is widely used in fluorescent probe design,chemical tissue monitoring of lipids,lipid and protein staining and other fields.However,the defects of Nile Red dye（high hydrophobicity,quantum yield easily affected by solvent polarity,difficulties in structure modifying,etc.）limit its further application and development in the field of fluorescent probes.Most of the probes reported have focused on identifying and differentiating objects at the cellular or tissue level,making it impossible to study their role in living systems and therefore the role of the analytes in physiological and pathological processes can not be understood.In contrast,in vivo imaging and the detection of the analytes in blood samples can solve this problem more intuitively and accurately.The reasons for this are as follows:First,in vivo imaging technology can be used to trace subtle changes in the course of the disease,and it can also achieve the advantages including non-invasive,in-situ detection,visualization and high spatial and temporal resolution.Secondly,the detection of the content of substances in blood samples is usually one of the commonly used methods for disease diagnosis in medicine.This is because the change of the content of certain substances in blood is often closely related to certain diseases and therefore the realization of the study of analytes in blood samples can provide effective guidance for clinical medicine.In order to achieve in vivo imaging and detection of analytes in blood samples,the probe itself needs to have extremely superior optical properties,first of all,the ability to perform strong biological sample penetration in living animals.The second is to reduce the interference of spontaneous fluorescence and absorption of biomolecules and other factors.Therefore,based on the above problems,a series of optical probes based on Nile Red platform were designed and applied to the detection of objects in vivo and blood samples.The details are as follows:（1）In this study,7-nitro-2,1,3-benoxadiazole（NBD）and 3-hydroxyl Nile Red（Nile-OH）was used as fluorescence quenching group and fluorescence reporter group respectively and therefore a NIR probe NN with excellent performance based on photoinduced electron transfer（PET）mechanism was developed.When the weakly fluorescent probe NN reacts with cysteine（Cys）,it will emit the green fluorescence（NBD-Cys）representing the product of cysteine and NBD group through rearrangement and the near infrared fluorescence（Nile-OH）.While probe NN only emits NIR fluorescence（Nile-OH）after reacting with glutathione/hydrogen sulfide（GSH/H₂S）.In addition,the probe has good selectivity,stability,low cytotoxicity and low detection limit.More importantly,the probe not only distinguished Cys and GSH/H₂S in vitro,but also distinguished Cys and GSH/H₂S in vivo for the first time.The in vivo differentiation of Cys and GSH/H₂S provides a basis for further exploration of the role of these mercaptans in biological systems.（2）In this study,we designed and synthesized the first fluorescence probe Nile-Tpa,which can be used to evaluate the dynamic changes of blood polarity in normal mice and circulating tumor mice.A novel Nile-Tpa fluorescent probe with intramolecular charge transfer（ICT）properties was conjugated with Nile Red and triphenylamine as solvent chromotropic dyes.Besides the long emission wavelength,high quantum yield,low toxicity,remarkable solvatochromic effect and good lipid droplet localization ability,the probe Nile-Tpa can also detect polarity at the cellular level and in vivo level.Most importantly,the fluorescence probe successfully detected attenuated blood polarity in a mouse model of circulating tumor for the first time,indicating that this novel probe can be a potential tool for the diagnosis of cancer metastasis and is expected to provide new ideas for the diagnosis of tumor metastasis.（3）The viscosity detection of blood samples has strict requirements on testing tools.As far as fluorescent probes are concerned,an ideal blood viscosity probe needs to be self-calibrating to ensure accurate capture of subtle changes in signal.At the same time,the probe should be able to capture viscosity signals while shielding the autofluorescence interference from living bodies and blood samples.To solve the above problems,we designed and synthesized a ratiometric fluorescent probe Nile-TPE based on TBET（Through bond energy Transfer）mechanism.This viscosity probe combines the conjugation of Nile Red and tetraphenylethylene（TPE）,endowing the probe an ocean of advantages including near infrared emission,ratiometric response,good lipid droplet（LDs）targeting ability,wide emission peak spacing,etc.Most importantly,in addition to achieving viscosity ratiometric imaging in live mice for the first time,the probe can also detect microviscosity ratiometric in blood samples in a nondestructive manner while shielding spontaneous fluorescence interference from blood samples.The study also showed for the first time that blood viscosity in peritonitis and folliculitis mice was approximately 1.51 and 1.21-1.27 times higher than before modeling,respectively.Therefore,the probe Nile-TPE has great potential in non-invasive clinical diagnosis of viscosity-related diseases.In conclusion,the research content of this thesis mainly takes Nile Red dye as the fluorescence platform.A series of fluorescence probes with excellent photophysical and chemical properties based on PET,ICT and TBET mechanism respectively,and their fluorescence changes involve Turn on,Turn off and ratiometric response were synthesized.These probes were applied to differentiate the active mercaptan or microenviroment in vivo or at blood level through reasonable design and modification of Nile Red platform.This series of fluorescent probes has been proved to have great practical application prospects by experiments:1)The process of Cys and GSH/H₂S differentiation in vivo lays a foundation for further exploration of the role of these mercaptan in biological system;2)The novel Nile-Tpa probe can be used as a potential tool for the diagnosis of cancer metastasis and is expected to provide new ideas for the diagnosis of cancer metastasis.3)Nile-TPE has great potential in non-invasive clinical diagnosis of viscosity related diseases.