In recent years,fluorescent chemical probes are considered to be one of the most effective ways for the detecting of biological species,such as ions/anions,small signal molecules and proteins,because of its high sensitivity and good selectivity,real-time detection and good biocompatibility,as well as simple and convenient to operate.Fluorescence probes,which achieve the qualitative or quantitative analysis of specific samples through observing or testing the change of the color or fluorescence signals,have been more and more widely used in Environmental Sciences,Life Sciences and Materials Science and so on.One-photon fluorescence probes,due to their shorter excitation wavelength and bad penetration depth,are difficult to be employed for detecting and tracking ions or molecules in living tissues and in vivo which are much thicker than in living cells.Nowadays,two-photon microscopy imaging(TPM)has become the leading imaging technology in biomedical researches for its remarkable advantages over one-photon microscopy(OPM),such as near infrared excitation,better spatial orientation ability,increased penetration depth,as well as the reduced photobleaching and photodamage.Besides,a ratiometric fluorescent probe,measuring the ratio of fluorescence intensities at two different emission wavelengths can eliminate the influence of environmental factors such as concentration,photobleaching and background interference(pH,viscosity,polarity,temperature,and so forth)to realize quantitative detection.The study and development of two-photon fluorescence probes with excellent optical signal,have attracted the attention of scientists and research workers,and become one of the most important research fields and direction.Pyrophosphate(P2O74-,PPi)is an important anion for many cellular processes,such as cellular ATP hydrolysis,DNA and RNA polymerizations and other metabolic processes.In addition,the concentration of PPi also has a lot to do with many diseases.Abnormal PPi levels can lead to vascular calcification,which results in severe medical conditions.Patients with calcium pyrophosphate dehydrate(CPPD)crystals and chondrocalcinosis have shown high PPi level in their synovial fluid.Therefore,the selective detection of PPi in aqueous solution,especially in biological tissues,is of great importance.Formaldehyde(FA),the simplest aldehyde,is well-known for a carcinogenic and mutagenic pollutant.The pollution of indoor or atmospheric air and food caused by formaldehyde can lead to dizziness,headache or nausea,memory loss,and even death.At the same time,it is also endogenously produced as a metabolic intermediate in most living organisms,wherein FA is formed during the metabolism of amino acids and xenobiotics by demethylase and oxidase enzymes.In a normal physiological brain,the FA has been proposed to play a role in the storage,preservation,and retrieval of long-term memory through DNA demethylation cycles.However,FA in excess of certain doses can cause many diseases,including Alzheimer’s disease,allergic lung inflammation,asthmatic symptoms,cardiovascular disease,and cancer.Therefore,selective and sensitive real-time detection methods of FA in environmental and biological system are highly needed.Based on reviewing a large amount of literatures and further studying the work of our group,we have designed two-photon fluorescence probes PC and MQAP to selectively recognise and detect PPi and FA respectively on the basis of coumarin and quinoline fluorophore.And the structure of the target compounds PC and MQAP was characterized by 1H NMR,13C NMR,and ESI-TOF MS spectroscopic methods.A series of important research were further studied successively,such as their optical properties,recognition mechanism and imaging in vivo.1.We developed a novel "on-off-on" two-photon fluorescent probe(PC)for Cu2+/PPi based on 7-substituted coumarin,which is a good two-photon fluorescent group that we previously developed.The 7-position of coumarin was decorated with 1-ethynyl-4-methoxybenzene to achieve more effective conjugation system and good two photon optical properties.The specified Cu2+-binding group was grafted to the coumarin system to achieve the selectivity for Cu2+ and exhibited an "on-off"fluorescent signal with the presence of Cu2+ in aqueous solution.In the meantime,the ensemble complex PCCu showed highly sensitive and selective for PPi.And PPi can snatch Cu2+ from the PCCu complex and recover the fluorescence of PC in aqueous solution and show "off-on" fluorescence responses.To the best of our knowledge,PCCu is the first two-photon fluorescent probe for detecting PPi in polymerase chain reactions(PCR),living cells,thick tissues and zebrafishes with lower toxicity and good biocompatibility.2.Herein,we developed an Aza-Cope Reactivity-Based ratiometric two-photon fluorescent probe(MQAP)for the detecting of FA based on 6-substituted quinolone,an excellent two-photon fluorescent group with large two-photon absorption cross section for cell imaging that was previously developed by our group.The homoallylamino group,as the reaction point for selectively responding to FA,was linked directly to the substituted quinoline skeleton.Upon reaction with FA,the homoallylamino group can react with FA to form an imine intermediate,which further undergoes subsequent 2-aza-Cope rearrangement and hydrolysis and finally yields an aldehyde changes into the aldehyde group with strong electron withdrawing ability.Then,the fluorescence emission wavelength of the probe might give rise to a red shift(85 nm)because the intramolecular charge transfer(ICT)process from the π-conjugatd electron donor(methoxy phenyl alkynyl)group to the aldehyde group enhances in the excited quinolone fluorophore.Selective ratiometric detection signal based on 2-aza-Cope rearrangement was confirmed by theoretical calculation,HPLC and MS spectra analysis.Moreover,the probe MQAP was also successfully applied to image FA in living cells and 5 days old zebrafish under TPM owing to its enlarged two-photon absorption cross-section and excellent biocompatibility. |