| Mitochondrion is an essential organelle within eukaryotic cells,utilizing oxygen to digest carbohydrates and fats,and releasing reactive oxygen species(ROS)as well as biochemical energy.Biothiols play important roles in regulating ROS homeostasis,which mainly occurs on mitochondria respiratory chain.Cysteine(Cys)and homocysteine(Hcy)in mitochondria are sensitive to the mitochondrial oxidative stress and further result in many diseases such as liver damage,weakness and Alzheimer's disease.Therefore,real-time monitoring of Cys and Hcy level in mitochondria is of great significance to uncover role of these amino acids in ROS homeostasis.Fluorescent probes have been evaluated as the most powerful tools to monitor cytosolic Cys/Hcy in biological system due to their excellent sensitivity and high selectivity.Unfortunately,most of reported Cys/Hcy fluorescent probes failed to target the mitochondria in living cells.To date,most of reported fluorescent probes are designed based on one-photon technology(OPM)with short excitation wavelength that limits their applications in deep-tissue and in vivo,owing to their faultiness,including photon-bleaching,photo-damage,cellular auto fluorescence as well as to the shallow penetration depth(<80?m).Recently,two-photon microscopy(TPM)has become the leading imaging technology in biology research for its obvious advantages over OPM,such as localized excitation,increased penetration depth,and the reduced photo-bleaching and photo-damage.Considering the inherent complexity and constant evolution of an organism,ratiometric measurement is superior to a single emission intensity measurement because it can eliminate most possible effects of environmental variations,probe distribution,and instrumental performance and thus offer a more accurate analysis.Therefore,based on reading a lot of literatures,we designed and synthesized a new two-photon fluorescence probe based on quinoline fluorescent group(Mito-MQ),which was used to detect cysteine and homocysteine in mitochondria.The main contents are as follows:We reported a novel ratiometric two-photon fluorescent probe(Mito-MQ).The aldehyde group was linked directly to the quinoline skeleton as the specific reaction point for Cys/Hcy.Triphenylphosphonium(TPP)group,which was widely used as the mitochondria targeting group,was connected to the fluorescent group to drive the probe to locate in mitochondria.Mito-MQ showed excellent two-photon fluorescence as a result of intramolecular charge transfer(ICT).We believe that the aldehyde group in Mito-MQ can form thiazolidine/thiazinane with Cys/Hcy through cyclization reaction.Mito-MQ showed the ratiometric fluorescent detection signal(the green-to-blue emissionfrom 517 nm to 460 nm)to cysteine(Cys)and homocysteine(Hey)over glutathione(GSH),along with the fast response rate(10 min).The-detection mechanism was illustrated by 1H-NMR,ESI-MS and theoretical calculation.The co-localization coefficient of 0.87 between Mito-MQ and MitoTracker Red revealed that the probe was predominantly present in mitochondria,therefore,Mito-MQ was successfully applied to detect mitochondrial oxidative stress by detecting the change of Cys/Hcy.Moreover,imaging in fresh tissue slices indicated that Mito-MQ could work in deep tissue(ca.130?m)under two-photon excitation.Furthermore,the measurement of Cys/Hcy detection in zebrafish showed that probe can be used in determination of biothiols in vivo. |
PDF Full Text Request