Construction And Application Of Long-wavelength Excitated And Solid-state Chemiluminescent Probes

At present,fluorescence imaging has become one of the most important methods of detecting biomolecules in living cells and in vivo.Combined with fluorescence imaging technique,small molecule fluorescent probes with simple design,easy structure modification and fast response have been widely used in biomedicine and environmental monitoring.However,traditional small molecule fluor escent probes still have many drawbacks,such as large autofluorescence interference,low tissue penetration depth and poor imaging resolution.Combined with two-photon,near-infrared and chemiluminescence imaging techniques,a series of small-molecule fluorescent probes with excellent performance were constructed and applied for the detection of various biomolecules.The details are as follows:(1)In Chapter 2,inspired by the activation of anti-malarial agents in living cells by labile heme(LH),we synthesized a two-photon LH-reactive small molecule fluorescent probe HNG.Traditional methods of heme detection cannot be used to distinguish between LH and protein-binding heme(protein-H).HNG could selectively recognize LH by utilizing the potential difference between LH and protein-H,and was highly sensitive to LH with a minimum detection concentration of 20 nM.In living cells,HNG could detect the changes of endogenous LH.However,the traditional method can only be used to determine the total amount o f heme in the cell extract,which greatly limits the application of the traditional method in LH-related diseases.Therefore,we believe that HNG is a powerful diagnostic tool for LH-related diseases.We investigated the ability of HNG to be used in the mice hemolysis model and evaluated the relationship between hemolytic condition with the damage of liver and LH content.We found that hemolytic condition was positively correlated with liver damage and LH content.Subsequently,we also performed two-photon imaging of liver tissue during hemolytic condition,and obtained nice imaging results.HNG enables detect LH in living cells and deep tissues,and provides an effective molecular design strategy for the development of LH-reactive probes for other purposes.(2)Two-photon imaging technique can not only be used to detect some small biological molecules,but also to analyze some biological macromolecules suc h as biological enzymes.Endoplasmic reticulum aminopeptidase 1(ERAP1)is an important biological enzyme found in the endoplasmic reticulum.There is currently no effective method for the detection of the activity of ERAP1 in living cells and biological tissues.In Chapter 3,we constructed a two-photon molecular fluorescent probe(SNCL)for the detection of intracellular ERAP1.This probe can monitor the change of ERAP1 content in endoplasmic reticulum under different redox condition.Meanwhile,SNCL can also be used for imaging the catalytic activity of ERAP1 in deep tissues.By comparing the effects of one and two-photon fluorescence imaging in the tissue,two-photon imaging provides a brighter image in deeper tissue than one-photon imaging.(3)Although the two-photon imaging technique solves the problems caused by short excitation wavelength,most two-photon probes have emission wavelengths below 600 nm,which makes it difficult for fluorescent signals to penetrate out of biological tissues.In chapter 4,combined with the advantages of near-infrared fluorescent probes,a cell membrane-targeted near-infrared small-molecule fluorescent probe ANRP was constructed to monitor the release of CO from living cells.A quaternary ammonium salt structure was attached to the hydrophobic Nile red dye for building a cell membrane targeting dye ANR.Using this design strategy,we also synthesized two other cell membrane targeting dyes.The ANR dye can be localized to the cell membrane rapidly(1 min)and can be anchored to the cell membrane for a long time.Then,ANR was coordinated with metal palladium to construct a cell membrane-targeted near-infrared CO molecular fluorescent probe ANRP.This probe has high selectivity and sensitivity to CO with a minimum detection limit of 0.23 ?M.In addition,the probe can be anchored to the cell membrane while monitoring the release of CO from living cells after exposure to different stimuli.Combined with the advantages of near-infrared imaging technique,the probe was successfully used for the detection of CO in vivo.(4)Chemiluminescence,which does not require additional excitation light source,can eliminate the interference caused by the excitation light source and improve imaging resolution,and is a promising imaging method.The probe based on solid-state fluorophore can be precipitated in situ after responding to the target,enabling in situ imaging of the target.However,the excitation wavelength of these probes is short.It is difficult to increase absorption wavelength of the dye to a large extent by structural modification such as expanding conjugation.In Chapter 5,we develop a new solid-state chemiluminescent probe HPQCL-Cys that can be used in vivo by combining solid-state luminescence with chemiluminescence.This probe showed high selectivity and reactivity to Cys.A large amount of particles are produced during the reaction of HPQCL-Cys to Cys.We separate the solid from the reaction and find that the particle luminescence is the main source of chemiluminescence,which fully proves the feasibility of the design strategy of solid-state chemiluminescent probe.In addition,we successfully applied HPQCL-Cys for the detection of Cys in vivo.Using these small molecule probes,the purpose of improving imaging resolution is achieved,and the detection of targets in deep tissues is performed.These probes provide effective methods for imaging other biomolecules.