Design And Synthesis Of New Probes For Chemiluminescence In Vivo Imaging And Exploration Of Small Animal Imaging

Recent years,chemiluminescence in vivo imaging has development,the related literature shows that more and more attention has payed to the rearch of chemiluminescence in vivo imaging.The lack of suitable for special probe in vivo analysis may become the biggest problem that constraint development of in vivo imaging.The primary problem of chemiluminescence development of in vivo imaging technology is the applied research in the design and synthesis of special imaging probe.Theses pecial probes should have the following characteristics:higher chemiluminescentous efficiency and good water solublity and emitting wavelength as close as possible to the in vivo optical window,good humoral stability and so on.we design and synthesis of a class of chemical luminescence probe,which is characterized by cyclic hydrazide structure as the group responsing to ractive oxygen species,fluorecein and rhodamin as the parent structure that bringing high fluorescence quantum yield to the probe.The chemiluminescent and fluorescent properties of the new probe is predicted through theoretical calculation and analysis.5,6-diformylhydrazinefluorescein will be well soluble by retaining polar groups of fluorescein.At the same time,the probe design should have good biological safety based on the knowledge of luminol and fluorescein.These characteristics are beneficial to chemiluminescence in vivo imaging.In chapter 1,the principle of CL and chemliminescence in-vivo imaging was introduced.The common CL probe and CL in-vivo imaging probe was also summarized.The research work of the dissertation was made up of two sections:(I)Synthesis of chemliminescence in-vivo imaging probe and its CL property study(1)Synthesis of 5,6-diformylhydrazinefluorecein.On the basis of summarizing synthetic method of fluorescein and luminol,organic synthesis of 5,6-diformylhydrazinefluorecein is presented and optimized.5,6-diformylhydrazinefluorecein is synthesized,separeted on the optimum conditions.Molecular structural is characterized by 1HNMR,13CNMR and MS.(2)CL property study of 5,6-diformylhydrazinefluorecein.The fluorescence spectrum,fluorescence efficiency,CL spectrum and CL efficiency are investigated separately.The results show that:? 5,6-diformylhydrazinefluorecein shows low fluorescence efficiency(6.01%),while its luminophor 5,6-dicarboxyfluorecein shows high fluorescence efficiency(89.9%);? pH has an effect on 5,6-dicarboxyfluorecein CL,CL efficiency drops rapidly with pH decreasing,compared to rising of CL efficiency relative to luminol.CL efficiency relative to luminol reaches maxmum when pH=7.4.? 5,6-diformylhydrazinefluorecein reacts with various ROS including H2O2?1O2?·O2-?HO·?ClO-on physiological conditions,and the corresponding CL efficiency are all higher than luminol;?The 5,6-diformylhydrazinefluorecein CL gives green light with wavelenth of 523 nm.These CL characteristics are benefit to in-vivo imaging analysis.(3)Synthesis and CL property study of 5,6-diformylhydrazinerhodamineB.5,6-diformylhydrazinerhodamineB is successfully synthesized using organic synthesis of 5,6-diformylhydrazinefluorecein for reference.5,6-diformylhydrazinerhodamineB CL gives orange light with wavelenth of 585 nm,the result show that organic synthesis of 5,6-diformylhydrazinefluorecein provide us a synthetic method of CL probe that wavelength can modulate depending on xanthene.When reacts with NaCIO,5,6-diformylhydrazinerhodamineB shows maxmum CL efficiency at pH=7.4.The relative CL efficiency of 5,6-diformylhydrazinerhodamineB reaches 240.(4)Synthesis and CL property study of 5,6-diformylhydrazinerhodamine110.5,6-diformylhydrazinerhodamine 110,as a Label CL reagent,is successfully synthesized using organic synthesis in(1).5,6-diformylhydrazinerhodamine110 CL gives green light with wavelenth of 530 nm.5,6-diformylhydrazinerhodaminell0 shows maxmum CL efficiency at pH=7.4 When reacts with NaClO which similar to 5,6-diformylhydrazinerhodamineB.Maxmum CL area reaches 1.5 fold of luminol.(?)Exploring the use of 5,6-diformylhydrazinefluorescein for small animal imaging.(1)5,6-diformylhydrazinefluorescein as a chemiluminescent probe applying on in vivo imaging of inflammation.The results show that in vivo CL imaging have good response to inflammation caused by muscle injury,bleeding gums,contact dermatitis.The in vivo CL imaging sensitivity of 5,6-diformylhydrazinefluorescein and luminiol is compared using a chemical contact dermatitis model,the results show that the 5,6-diformylhydrazinefluorescein is more sensitive when detecting chemical contact dermatitis.(2)5,6-diformylhydrazinefluoresceim is used to catalyst labelled CL imaging.5,6-diformylhydrazinefluorescein react with H2O2 give weak CL,while reaction kinetics curve is very flat,horseradish peroxidase(HRP)can act as a catalyst and enhance the CL intensity.Based on above results,HRP and HRP HRP-labeled Goat Anti-Mouse IgG is investigated by 5,6-diformylhydrazinefluorescein CL imaging.Potential application of the CL imaging besides ROS detection is explored.The results show 5,6-diformylhydrazinefluorescein can realize the detection of trace HRP in nude mouse.