The Design,Synthesis And Application Of Long-wavelength Emissive Two-photon Fluorescent Probe For Nitric Oxide

Two-photon excited fluorescence is the process that molecule absorbs two identical NIR photons to excited the flourophore and produce fluorescence,which featured with NIR light excited wavelength(700-900 nm)and localized excitation only at the focus.Compared with one-photon fluorescence microscopy,two-photon microscopy(TPlM)possesses several advantages,such as deeper imaging depth,less photobleaching and higher spatial resolution.Recent years,two-photon microscopy has been widely applied to the research areas of biology and medicine.Currently available two-photon fluorophores including naphthalene derivatives,fluorine derivatives,naphthalimide derivatives,fluorescein and rhodamine derivative featured with NIR(700-900 nm)excitation and short-wavelength(<600 nm)emission.As is known to all,fluorescence imaging includes fluorescence excitation and fluorescence emission.During the microscopy of complicated biological samples,the longer excitation and emission wavelength will cause less light-scattering and back ground fluorescence.The negligible light scattering and background fluorescence will contribute the image depth.The NIR excitation under two-photon model is suitable for long-wavelength excitation,but short emission wavelength still hampered the application of two-photon microscopy.So far,red/NIR emissive two-photon probe has been rarely reported.Therefore,developing long-wavelength emissive two-photon fluorophore and probe is quite important for two-photon microscopy.This dissertation was focused on the fundamental issue research and conducted the PhD's research by the virtue of the basic principles of two-photon absorption and probe design.The main contents of the dissertation are presented as follows.1)Designing and synthesizing five dyes.By introducing heteroatom to the ?-system,designing excited state intramolecular proton transfer and enlarging the ?-conjugate plane,quinolone derivative,naphthalene derivative,benzo[g]coumarin derivative,Nile Red derivative and silicon-rhodamine dyes were obtained,respectively.Studying the influences of molecular polarity,electron donor and acceptor and the ?-conjugated system on the maximal emission wavelength and two-photon asbsorption properties,and three long-wavelength emissive two-photon fluorophore were preliminary obtained.2)A benzo[g]cumarin-based far-red emissive two-photon probe NCNO for NO was exploited,and the recognition of NO is based on the N-nitrosation reaction of secondary aromatic amine and NO.The detection of NO relied on the PET mechanism.O-phenylenediamine can easily react with dehydroascorbic acid and methylglyoxal by condensation reaction,and also can be oxidized by reactive oxygen species.For these reasons,o-phenylenediamine-based probe usually shows poor selectivity for NO.Due to N-nitrosation reaction is a specific reaction for the secondary aromatic amine and NO,NCNO show a high selectivity for NO among reactive carbon species(ascorbic acid,dehydroascorbic acid,methylglyoxal,etc.),ROS,RNS and metal ions.The two-photon excited background fluorescence emissions collected at different channels were also investigated in the tissue imaging,the results confirmed that probe with red-light emission have much lower background fluorescence interference.At last,the probe was applied in the detection of NO in mice renal ischemia-reperfusion injury model for the first time.3)A Nile red-based far-red emissive two-photon fluorescent probe NRNO for NO was developed.In the probe NRNO,Nile Red derivate was chosen as two-photon fluorophore and o-phenylenediamine was selected as NO recognition group.The maximal emission wavelength of Nile Red derivate is 650 nm due to the large rigid conjugate plane and polarity.The probe can be employed in the detection of cellular exogenous and endogenous NO.Owing to the far-red emission and excellent two-photon absorption properties,the probe can be ultilized in the NO detection in deep tissues(maximal imaging depth 170?m).Ultimately,the probe was applied in the in situ NO detection in a LPS-induced mice tissue inflammation model.4)The two-photon absorption property of the NIR emissive dye silicon-rhodamine(SiR-OMe)was first studied,and a new NIR emissive two-photon NO probe SiRNO was developed.The fluorophore and recoginition grounp was covalently linked by carbon carbon single bond,which greatly improves the quenching efficiency of PET.After the completed reaction of the probe and NO,the probe SiRNO exhibited 80-fold fluorescence enhancement.The calculated limit of detection is 14 nM.The results indicated the probe show an ultrahigh sensitivity toward NO.The probe both can sensitively detect exogenous and endogenous NO in cell.At last,the probe can detect NO in tumor tissues by two-photon microscopy.