Constructin Of Two-photon Fluorescent Probes With Long-wavelength Emission For Reactive Oxygen Species And Their Applications In Bioimaging

In recent years,fluorescence imaging technology has been widely used,and it is a powerful tool to observe cell morphology and structure and study the physiological or pathological process of cells.The development of two-photon fluorescence imaging technology provides a good theoretical basis and technical means to overcome the defects of single-photon fluorescence imaging technology.The two-photon fluorescence imaging technology has the advantages inccluding better three-dimensional spatial positioning ability,deeper tissue penetration,lower interference from background fluorescence,which can reduce the light damage to biological samples and the photobleaching effect of fluorescent molecules during imaging,and is conducive to long-term and deep-level observation of biological dynamic information in cell,tissue,and animals.With the advantages of two-photon fluorescence imaging technology,researchers developed a series of two-photon fluorescent probes.However,these two-photon fluorescent probes only solve the problem of short-wavelength excitation of single-photon probe,and there are still defects caused by the emission wavelength in the short wavelength region（usually<550 nm）.Some endogenous molecules（folic acid,riboflavin,NADH）in vivo can also be excited by two-photon laser,which will emit short-wavelength emission light,thus generating background fluorescence interference to the above probes.At the same time,the short-wavelength emission of the probe will also be absorbed by other molecules in vivo（hemoglobin and oxyhemoglobin）,thus reducing the intensity of the original fluorescence signal in the imaging.Therefore,it is of great significance to develop a two-photon fluorescent probe with long-wavelength emission.Compared with the two-photon probe with short-wavelength emission,the two-photon probe with long-wavelength emission has the advantages of original emission that is less absorbed by biomolecules,deeper tissue penetration depth,less background fluorescence interference,and has better performances in imaging.Based on the above reasons,two kinds of two-photon fluorescent probes with long-wavelength emission are designed and synthesized for two kinds of active oxygen（hypochlorite and peroxynitrite）in this paper.The contents are as follows:（1）In the second chapter,we developed a mitochondria-targetable two-photon fluorescent probe（Mito-TP-Cl O）with a far-red to near-infrared emission for sensing hypochlorite.The probe consists of two parts:mitochondria-targetable group（a triphenylphosphonium cation）and fluorescent group with the structure that has a specific response for Cl O^-.Mito-TP-Cl O showed a high sensitivity and a high selectivity to Cl O^-,with a linear response range of 6.0×10^-8 to 1.0×10^-5M and a detection limit of 25 n M.The product of response of Mito TP clo to hypochlorite has a large two-photon absorption cross-section（267 GM）and excellent photostability,which resulted in low interference from background fluorescence and a large imaging depth（230μm）during detecting hypochlorite in vivo.In addition,Mito-TP-Cl O has been successfully applied to the detection of endogenous Cl O^-in bacteria-infected cells and inflammatory mouse model.These results show that the probe Mito-TP-Cl O is a powerful tool to monitor the concentration of Cl O^-in mitochondria and to study the role of Cl O^-in mitochondria.（2）Drug-induced liver injury has become a hot topic in the field of public health because of its difficult to monitor and serious harm to public health.Peroxynitrite（ONOO^-）is a kind of reactive oxygen species（ROS）,which can be used as a biomarker for the evaluation and diagnosis of drug-induced liver injury.In the third chapter,by expandingπ-conjugated system of traditional rhodamine,a two-photon excited near-infrared fluorescent probe（NIR-ONOO）was developed to realize the diagnosis and monitoring of drug-induced liver injury in cells.NIR-ONOO has a high sensitivity and a selectivity to ONOO^-,with a linear response range of 5.0×10^-8 to 1.0×10^-5 M,and a detection limit of 15 n M.At the same time,NIR-ONOO has been successfully applied to the detection of endogenous and exogenous ONOO^-,and also to the detection of ONOO^-in tissue and inflammatory mouse model.Finally,we applied the probe NIR-ONOO to realize the imaging monitoring of ONOO^-in the process of drug-induced liver cell injury and its remediation.The above results show that the probe NIR-ONOO is a powerful tool for imaging ONOO^-in vivo,which provides technical support for the evaluation and monitoring of drug-induced liver injury.