Construction And Application Of ICT Two-photon Fluorescent Probes For Organelles

In living cells,each organelle complements each other to ensure normal life and physiological activity and is the basic unit for the normal operation of cells.More importantly,the various bio-enzymes on the organelles,such as lysosomal hydrolase,mitochondrial ATPase and the ?-glutamyl transpeptidase(GGT)are the basic functional molecules which maintain the normal operation of all kinds of organelles.Therefore,real-time in situ analysis of activity changes of bio-enzymes on the organelles has become an important entry point for the study of changes in organelle biological function.Two-photon fluorescence probe has become an important tool to study the activity of organelle's bio-enzymes based on the high spatial,temporal resolution,excellent penetrating ability and "zero" interference.Thus,we designed three ICT-based two-photon fluorescence probes targeting organelles and studied the real-time in situ analysis property of biological activity of the relevant probes at the cell,tissue and in vivo level.Lysosome contains more than 60 kinds of acid hydrolases and is an important organelle that has the function of digestion in living cells.Due to the activity of various hydrolytic enzymes,the lysosomal polarity has become an important microenvironmental parameter in living cells.It is not only involved in many of the molecular events in cells,but also closely related to apoptosis and death.Thus,an intramolecular charge transfer(ICT)two-photon ratiometric fluorescent probe(DC)was designed and synthesized based on the derivative of flunarizine.DC appeared different fluorescence intensities at 580 nm and 600 nm,and its fluorescence change presented a linear change on ratio mode in the range of polarity(?f = 0.2230-0.3274).The lowest detection of DC for polarity change can be as low as ?f = 0.018.More importantly,DC can real-time estimate cancer cell apoptosis through ultrasensitive detection of lysosomal micro-polarity by microscopic imaging and flow cytometry.Mitochondrial adenosine triphosphatase(ATPase)not only participates in maintaining the balance of energy metabolism in the body,but also has a great relationship with cell apoptosis.Based on this,an intramolecular charge transfer(ICT)two-photon ratiometric fluorescent probe(OPM)was designed.The probe can not only detect the mitochondrial ATPase activity(the detection limit of 15.77 U),but also generate the ratio signal(I590 nm / I565 nm)at 1.0 s,and achieved in situ monitoring and imaging of themitochondrial ATPase activity in cancer cells,but also can real-time monitor and imagine mitochondrial ATPase activity during cancer cells apoptosis.?-Glutamyl transpeptidase(GGT)is an important marker of cancer diagnosis and its effect on?-glutamyl compounds,with optical and stereospecific transfer catalytic activity.At present,there is still controversy over whether the GGT on the ER of intracellular GGT has the catalytic activity.Thus,an ER-targeted GGT-specific real-time and in-situ detection two-photon fluorescent probe(SCP-glu)was designed.The fluorescence intensity of this probe at 425 nm showed a good linear relationship with the GGT enzyme activity.And based on excellent biological characteristics,such as proper water oil amphiphism,good photostability,the probe can be used to the real-time and in situ monitoring and imaging analysis of GGT enzyme activity in ER of living cells and in vivo.