Synthesis Of Near-infrared Excited Fluorescent Probe And Bioimaging For Detecting The Bioactive Molecule

Fluorescent molecular imaging has advanced drastically over the past decade.With the development of high-resolution microscopy techniques and the ability to visualize intracellular molecular events,there is a growing need for new fluorophores to accompany these fast-developing techniques.Therefore,there has been substantial development of alternative fluorophores for single-molecule detection and molecular imaging.These rationally designed fluorophores have infinite possibilities and novel fluorophores are constantly being produced for different applications.Fluorescent probes have become powerful tools in biosensing and bioimaging because of their high sensitivity,specificity,fast response,and technical simplicity.In the last decades,researchers have made remarkable progress in developing fluorescent probes that respond to changes in microenvironments（e.g.,pH,viscosity,and polarity）or quantities of biomolecules of interest（e.g.,ions,reactive oxygen species,and enzymes）.Although wide applications have been made in fluorescence probes,several defect of conventional fluorescent probes,such as photobleaching problem,autofluorescence interference in cells and tissues and shallow penetration depth,which are still restricted their application in biological samples.However,two-photon excited（TPE）fluorescent probe based bioimaging can obtain improved three-dimensional spatial localization and increased imaging depth,as well as reduce phototoxicity or photodamage to biological samples,more and more attentions have been paid to TP fluorescence probe.In this paper,we designed a series of fluorescence probe based TPE for detecting the small biological molecules,and we have demonstrated the feasibility and practicability of probes bioimaging and the adhibition of TP imaging in the living cells.Details are as follows:（1）A ratiometric TP fluorescent probe was designed by modifying a ICT-based two-photon fluorescent dye（4-hydroxy-1,8-naphthalimide）with a cyanate（RO-CN）as an H₂S specific recognition unit.In the present of H₂S,with the formation of thiocarbamates and it could be rapidly hydrolyses to its hydroxyl derivative in aqueous environments,the cyanate（RO-CN）will be removed and the TP fluorophore will be relased.The probe avoids the influence of background fluorescence and displays remarkable fluorescence intensity ratio(F_{552 nm}/F_{448 nm})enhancement in presence of H₂S,which exhibited a high sensitivity to H₂S with a detection limit of 0.24μΜas well as relatively quick response time（<90 s）.Probe 1 possesses distinguished selectivity over other cations/anions and biologically active compounds.In addition,Probe 1 could accurately detect H₂S in presence of other biologically related species.Probe 1 was then successfully applied in imaging of exogenous and endogenous H₂S in Hela cells.And also confirmed that Probe 1 has the capability of TP imaging for detecting H₂S in the living cells.All of these features make it an accessible tool to practical applications in biological systems.（2）A FRET-based ratiometric TP fluorescent probe TPR-O was developed for detecting O₂^·-.Firstly,a FRET-based ratiometric two-photon fluorescent platform was synthesed,Np-Rhod.This platform was assembled by directly connecting a D-π-A structure two-photon naphthalene derivative with a rhodol fluorophore via the FRET strategy.TPR-O was designed by introducing a trifluoromethanesulfonate group as recognition unit for O₂^·-to a TP-FRET cassette.In the absence of O₂^·-,the acceptor exists in a nonfluorescent lactone form,the FRET remains off,and TPR-O shows a low fluorescence intensity ratio(F_{541 nm}/F_{448 nm}).When O₂^·-attacks the recognition group of TPR-O,the TP-FRET cassette is released and FRET is initiated,resulting in a high fluorescence intensity ratio(F_{541 nm}/F_{448 nm}).TPR-O exhibits high selectivety for O₂^·-,with a detection limit of 6.2 nM.More importantly,TPR-O may be used to detect O₂^·-in living cells.